Alleviating oxidative stress disorders with pufa derivatives

ABSTRACT

Some aspects of the invention provide for essential fatty acids which are substituted in specific positions to slow down oxidative damage by Reactive Oxygen Species (ROS), and to suppress the rate of consequent formation of reactive products, for the purpose of preventing or reducing the damage associated with oxidative stress associated diseases such as neurological diseases and age-related macular degeneration (AMD).

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. application Ser. No.12/916,347, filed Oct. 29, 2010, which claims priority to U.S.Provisional Application No. 61/256,815, filed Oct. 30, 2009, both ofwhich are hereby incorporated herein by reference in their entireties.

BACKGROUND Field

Isotopically modified polyunsaturated fatty acids (PUFAs) and othermodified PUFAs are useful in methods of treating certain diseases.

Description of the Related Art

U.S. application Ser. No. 12/281,957 assigned to the same assignees asthe present application, refers to a class of compounds that, wheningested, result in the formation of bodily constituents, for example,fats that are functionally equivalent to normal bodily constituents butwhich have a greater resistance to degradative/detrimental processessuch as those mediated by reactive oxygen species (ROS), reactivenitrogen species (RNS) or radiation. This application, which isincorporated herein by reference, refers to an essential nutrient inwhich at least one exchangeable H atom is ²H and/or at least one C atomis ¹³C. This application also discloses 11, 11 dideutero linoleic acid.

11, 11 dideutero linoleic acid and 11, 11, 14, 14 D4 linolenic acid andsimilar compounds wherein the C atom in the deuterated methylene groupmay be ¹³C is disclosed. Shchepinov, M, Reactive Oxygen Species, IsotopeEffect, Essential Nutrients, and Enhanced Longevity, RejuvenationResearch, vol. 10, no. 1, (2007). This article is incorporated herein byreference.

Although oxidative stress may be associated with various diseases, it isunpredictable which antioxidants will be successful in treating variousdiseases. Thus, there is a need in the art for successful treatment forvarious diseases. Therefore, there is a need in the art for additionalisotopically modified polyunsaturated fatty acids (PUFAs) and othermodified PUFAs useful for treating various diseases.

Replacing certain positions of PUFAs may also prevent or slow thehelpful metabolic processes in which PUFAs are involved, and thus itwould be helpful to the art to determine modified PUFAs that willsufficiently maintain these metabolic processes while resistingdetrimental oxidative processes.

It would also be helpful to the art to determine the minimum amount ofheavy atoms substitution necessary to prevent detrimental oxidativeprocesses to save costs on heavy atom substitution. These and otheraspects are addressed herein.

SUMMARY

The present disclosure addresses these needs and the need for additionalisotopically modified polyunsaturated fatty acids (PUFAs), mimetic orester pro-drug thereof. Further, present disclosure addresses the needfor new methods of treating and preventing specific diseases usingmodified PUFAs in subjects such as human subjects.

Some embodiments include a method of treating or preventing theprogression of a neurodegenerative disease comprising selecting asubject that has a neurodegenerative disease or is susceptible to aneurodegenerative disease; administering an effective amount ofisotopically modified polyunsaturated fatty acid, mimetic or esterpro-drug thereof to the subject; wherein upon administration, theisotopically modified polyunsaturated fatty acid, mimetic or esterpro-drug thereof is incorporated in brain and/or neuronal tissue of thesubject. The patient who has a neurodegenerative disease may include asubject with a) Alzheimer's disease or is susceptible to Alzheimer'sdisease; b) has mild cognitive impairment or is susceptible to mildcognitive impairment; c) has Parkinson's disease or is susceptible toParkinson's disease; d) has schizophrenia or is susceptible toschizophrenia; e) has a bipolar disorder or is susceptible to a bipolardisorder; f) has amyotrophic lateral sclerosis or is susceptible toamyotrophic lateral sclerosis, among other diseases.

Some embodiments include a method of treating or preventing theprogression of an oxidative disease of the eye comprising selecting asubject that has an oxidative disease of the eye or is susceptible to anoxidative disease of the eye; administering an effective amount of atleast one isotopically modified polyunsaturated fatty acid, mimetic orester pro-drug thereof to the subject; wherein upon administration, theisotopically modified polyunsaturated fatty acid, mimetic or esterpro-drug thereof is incorporated in eye tissue of the subject. Thesubject with oxidative disease of the eye may include a subject havingretinal disease or is susceptible to a retinal disease, having agerelated macular degeneration or is susceptible to age related maculardegeneration, having diabetic retinopathy or is susceptible to diabeticretinopathy, or having retinitis pigmentosa or is susceptible toretinitis pigmentosa, among other diseases.

Additional embodiments include a method comprising selecting a subjectin need of increased levels of high-density lipoprotein and/or decreasedlevels of low-density lipoprotein; administering an effective amount ofisotopically modified polyunsaturated fatty acid, mimetic or esterpro-drug thereof to the subject; and wherein upon administration, thelevel of high-density lipoprotein is increased and/or the level oflow-density lipoprotein is decreased. Subjects may include those withatherosclerotic vascular disease or susceptible to atheroscleroticvascular disease, among other diseases.

Further embodiments include a method of treating or preventing theprogression of a mitochondrial deficiency or mitochondrial respirationdeficiency disease, such as a Coenzyme Q10 deficiency, comprisingselecting a subject that has a mitochondrial deficiency or mitochondrialrespiration deficiency diseases such as a Coenzyme Q10 deficiency or issusceptible to mitochondrial deficiency or mitochondrial respirationdeficiency disease comprising administering an effective amount ofisotopically modified polyunsaturated fatty acid, mimetic or esterpro-drug thereof to the subject; wherein upon administration, theisotopically modified polyunsaturated fatty acid, mimetic or esterpro-drug thereof is incorporated in mitochondrial membrane of thesubject. Subjects having other mitochondrial deficiency or mitochondrialrespiration deficiency diseases include a) nervous system disease or issusceptible to a nervous system disease, b) dyskinesia or is susceptibleto dyskinesia, c) ataxia or is susceptible to ataxia, d) musculoskeletaldisease or is susceptible to a musculoskeletal disease, e) muscleweakness or is susceptible to muscle weakness, f) a neuromusculardisease or is susceptible to a neuromuscular disease, or g) a metabolicdisease or is susceptible to a metabolic disease.

Methods also include a method of treating an inborn error of metabolismcomprising selecting a subject that has an inborn error of metabolism,administering an effective amount of isotopically modifiedpolyunsaturated fatty acid, mimetic or ester pro-drug thereof to thesubject; wherein upon administration, the isotopically modifiedpolyunsaturated fatty acid, mimetic or ester pro-drug thereof isincorporated in brain and/or neuronal tissue of the subject. The inbornerror of metabolism may be Down's syndrome, for example.

In some embodiments, a method comprises administering to a subject asufficient amount of an isotopically modified PUFA, wherein a cell ortissue of the subject maintains a sufficient concentration ofisotopically modified PUFAs to maintain autooxidation of the PUFAs.

Compounds and compositions are also contemplated such as apolyunsaturated fatty acid composition comprising an isotopicallymodified polyunsaturated fatty acid, mimetic or ester pro-drug thereofcomprising at least one ¹³C or at least two deuterium atoms at abis-allylic position, or a mimetic or mimetic ester thereof, wherein thecomposition is suitable for human consumption, wherein the isotopicallymodified polyunsaturated fatty acid or ester thereof or mimetic ormimetic ester thereof is capable of retaining its chemical identity whenincorporated in a bodily constituent of the subject following ingestionor uptake by the subject, or is capable of conversion into higherhomolog of the polyunsaturated fatty acid or mimetic thereof in thesubject; wherein the amount of isotopes in the isotopically modifiedpolyunsaturated fatty acid is above the naturally-occurring abundancelevel; and with the proviso wherein the isotopically modifiedpolyunsaturated fatty acid is not 11, 11, 14, 14, D4-linolenic acid or11, 11, D2-linoleic acid. The isotopically modified polyunsaturatedfatty acid or mimetic thereof may be an isotopically modifiedpolyunsaturated fatty acid selected from the group consisting of 11, 11,14, 14, D4-linoleic acid, 11, 11, D2-linolenic acid, and 14, 14,D2-linolenic acid. The isotopically modified polyunsaturated fatty acid,mimetic or ester pro-drug thereof may be an isotopically modifiedpolyunsaturated fatty acid further comprising deuterium at apro-bis-allylic position. The isotopically modified polyunsaturatedfatty acid, mimetic or ester pro-drug thereof may be a mimetic selectedfrom the group consisting of

or an ester pro-drug thereof. In some embodiments, these compounds andcompositions may be used for treating any of the diseases or disordersdisclosed herein.

The isotopically modified polyunsaturated fatty acid or ester pro-drugthereof may be an isotopically modified polyunsaturated fatty acid orester that has an isotopic purity of from about 50%-99%.

In other aspects, a polyunsaturated fatty acid composition comprises anaturally occurring polyunsaturated fatty acid, mimetic, or esterpro-drug thereof, that are modified chemically to be effective atpreventing specific disease mechanisms; wherein the chemicalmodification does not change the elemental composition of the naturallyoccurring polyunsaturated fatty acid, mimetic, or ester pro-drugthereof, with the proviso wherein the isotopically modifiedpolyunsaturated fatty acid is not 11, 11, 14, 14, D4-linolenic acid or11, 11, D2-linoleic acid. For example, the naturally occurringpolyunsaturated fatty acid, mimetic, or ester pro-drug may be stabilizedagainst oxidation, such as at oxidation sensitive loci. In some casesthe stabilization is through heavy isotope substitution. The oxidationsensitive loci may include substitution at the bis-allylic carbonhydrogen atoms.

These and other embodiments are described herein in more detail.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1. (A) ROS-driven oxidation of PUFAs; (B) formation of toxiccarbonyl compounds.

FIG. 2. ¹H- and ¹³C-NMR analysis of deuterated PUFAs described inExamples 1-4.

FIG. 3. Sensitivity of coq null mutants to treatment with linolenic acidis abrogated by isotope-reinforcement. Yeast coq3, coq7 and coq9 nullmutants were prepared in the W303 yeast genetic background (WT). Yeaststrains were grown in YPD medium (1% Bacto-yeast extract, 2%Bacto-peptone, 2% dextrose) and harvested while in log phase growth(OD_(600 nm)=0.1-1.0). Cells were washed twice with sterile water andresuspended in phosphate buffer (0.10 M sodium phosphate, pH 6.2, 0.2%dextrose) to an OD_(600 nm)=0.2. Samples were removed and 1:5 serialdilutions starting at 0.20 OD/ml were plated on YPD plate medium, toprovide a zero time untreated control (shown in top left panel). Thedesignated fatty acids were added to 200 uM final concentration to 20 mlof yeast in phosphate buffer. At 2 h, 4 h, and 16 h samples wereremoved, 1:5 serial dilutions prepared, and spotted onto YPD platemedium. Pictures were taken after 2 days of growth at 30° C. This panelis representative of two independent assays, performed on differentdays.

FIG. 4. Yeast coq mutants treated with isotope-reinforced D4-linolenicacid are resistant to PUFA-mediated cell killing. The fatty acidsensitive assay was performed as described in FIG. 6-1, except that 100ul aliquots were removed at 1, 2, and 4 h and, following dilution,spread onto YPD plates. Pictures were taken after 2 to 2.5 days, and thenumber of colonies counted. Yeast strains include Wild type (circles),atp2 (triangles), or coq3 (squares); Fatty acid treatments include oleicC18:1 (solid line), linolenic, C18:3, n-3 (dashed line) or11,11,14,14-D4-linolenic, C18:3, n-3, (dotted line).

FIG. 5. Separation and detection of fatty acid methyl ester (FAME)standards by GC-MS. FAMEs were prepared as described (Moss C W, LambertM A, Merwin W H. Appl. Microbiol. 1974; 1, 80-85), and the indicatedamounts of free fatty acids and 200 μg of C17:0 (an internal standard)were subjected to methylation and extraction. Samples analyses wereperformed on an Agilent 6890-6975 GC-MS with a DB-wax column (0.25 mm×30m×0.25-m film thickness) (Agilent, catalog 122-7031).

FIG. 6. Uptake of exogenously supplied fatty acids by yeast. WT (W303)yeast were harvested at log phase and incubated in the presence of 200μM of the designated fatty acid for either 0 or 4 h. Yeast cells wereharvested, washed twice with sterile water and then subjected toalkaline methanolysis and saponification, and lipid extraction asdescribed (Moss C W, Lambert M A, Merwin W H. Appl. Microbiol. 1974; 1,80-85; (Shaw, 1953 Shaw, W. H. C.; Jefferies, J. P. Determination ofergosterol in yeast. Anal Chem 25:1130; 1953). Each designated fattyacid is given as μg per OD_(600 nm) yeast, and was corrected for therecovery of the C17:0 internal standard.

FIG. 7. Chromatograms of the yeast extracts subjected to GC-MS analyses.The different traces represent the 0 and 4 h incubations, respectively.The peak area of Each FAME (C18:1, C18:3 and D4-linolenic) was dividedby the peak area of the C17:0 standard, quantified with a calibrationcurve. The endogenous 16:0 and 16:1 change very little, while theexogenously added fatty acids increased significantly.

FIG. 8. Survival of H- and D-PUFA treated MVEC cells after acuteintoxication by paraquat. For all cell types tested, D-PUFA hadprotective effect compared to controls, similar to that shown on Figurefor MVEC cells.

FIG. 9. D-PUFA partially attenuates MPTP-induced striatal dopaminedepletion in C57BL/6 mice. Mice, aged 8 weeks, were fed fat-free dietsupplemented with either D-PUFAs or H-PUFAs for 6 days, exposed to 40mg/kg MPTP, i.p., or saline, continued on D- or H-PUFA diet andsacrificed 6 days later. Striatal dopamine was measured by HPLC. MPTPproduced a robust depletion in H-PUFA-fed mice (78%) which wassignificantly less in the D-PUFA-fed cohort (47%).

FIG. 10. D-PUFA partially attenuates MPTP-induced nigral a-synaccumulation in C57BL/6 mice. Mice, aged 8 weeks, were fed fat-free dietsupplemented with either D-PUFAs or H-PUFAs for 6 days, exposed to 40mg/kg MPTP, i.p., or saline, continued on D- or H-PUFA diet andsacrificed 6 days later. Immunoreactivity for a-syn was observed insections from the substantia nigra of the cohorts. While neuropilstaining was apparent in both saline-treated groups, robust cell bodystaining was noted in H-PUFA-fed, MPTP-treated mice. An apparentreduction in the intensity and number of a-syn-positive cell bodies wasobserved in the D-PUFA-fed, MPTP-treated cohort by comparison. Bar=25μm.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT

As an introduction, lipid-forming fatty acids are well-known as one ofthe major components of living cells. As such, they participate innumerous metabolic pathways, and play an important role in a variety ofpathologies. Essential Polyunsaturated Fatty Acids (PUFAs) are animportant sub-class of fatty acids. An essential nutrient is a foodcomponent that directly, or via conversion, serves an essentialbiological function and which is not produced endogenously or in largeenough amounts to cover the requirements. For homeothermic animals, thetwo rigorously essential PUFAs are linoleic(cis,cis-9,12-Octadecadienoic acid; (9Z,12Z)-9,12-Octadecadienoic acid;LA; 18:2;n-6) and alpha-linolenic (cis,cis,cis-9,12,15-Octadecatrienoicacid; (9Z,12Z,15Z)-9,12,15-Octadecatrienoic acid; ALA; 18:3;n-3) acids,formerly known as vitamin F (Cunnane S C. Progress in Lipid Research2003; 42:544-568). LA, by further enzymatic desaturation and elongation,is converted into higher n-6 PUFAs such as arachidonic (AA; 20:4;n-6)acid; whereas ALA gives rise to a higher n-3 series, including, but notlimited to, eicosapentaenoic acid (EPA; 20:5;n-3) and docosahexaenoic(DHA; 22:6;n-3) acid (Goyens P L. et al. Am. J. Clin. Nutr. 2006;84:44-53). Because of the essential nature of PUFAs or PUFA precursors,there are many instances of their deficiency. These are often linked tomedical conditions. Many PUFA supplements are availableover-the-counter, with proven efficiency against certain ailments (Forexample, U.S. Pat. No. 7,271,315, U.S. Pat. No. 7,381,558).

Brain tissue is particularly rich in PUFAs, which constitute 35% of thephospholipids in the neuronal membranes of the brain (Hamilton J A. etal. J. Mol. Neurosci. 2007; 33:2-11). Three particularly important fattyacids, which are abundant in neuronal membranes, are: LA, which makes upcardiolipin; DHA, deficiencies of which can impede brain development andcompromise optimal brain function; and AA, which yields essential, butpotentially toxic, metabolic products.

PUFAs endow membranes, in particular mitochondrial membranes, withappropriate fluidity necessary for optimal oxidative phosphorylationperformance. PUFAs also play an important role in initiation andpropagation of the oxidative stress. PUFAs react with ROS through achain reaction that amplifies an original event (Sun M, Salomon R G, J.Am. Chem. Soc. 2004; 126:5699-5708). Of particular importance is amitochondrial membrane-specific PUFA-rich phospholipid cardiolipin,vital for electron transport Complex I activity (Paradies G, et al. Gene2002; 86:135-141).

Non-enzymatic formation of high levels of lipid hydroperoxides is knownto result in several detrimental changes. It negatively affects thefluidity and permeability of the membranes; leads to oxidation ofmembrane proteins; and these hydroperoxides can be converted into alarge number of highly reactive carbonyl compounds. The latter includereactive species such as acrolein, malonic dialdehyde, glyoxal,methylglyoxal, etc (Negre-Salvayre A, et al. Brit. J. Pharmacol. 2008;153:6-20). But the most prominent products of PUFAs oxidation are alpha,beta-unsaturated aldehydes 4-hydroxynon-2-enal (4-HNE; formed from n-6PUFAs like LA or AA), 4-hydroxyhex-2-enal (4-HHE; formed from n-3 PUFAslike ALA or DHA), and corresponding ketoaldehydes (Esterfbauer H, et al.Free Rad. Biol. Med. 1991; 11:81-128; Long E K, Picklo M J. Free RadBiol. Med. 2010; 49:1-8). These reactive carbonyls cross-link(bio)molecules through Michael addition or Schiff base formationpathways, and have been implicated in a large number of pathologicalprocesses, age-related and oxidative stress-related conditions andaging. Importantly, in some cases, PUFAs appear to oxidize at specificsites because methylene groups of 1,4-diene systems (the bis-allylicposition) are substantially less stable to ROS, and to enzymes such ascyclogenases and lipoxygenases than allylic methylenes.

There are many diseases that are oxidative stress-related, including,but not limited to, neurological diseases, diabetes, diseases associatedwith elevated concentration of low density lipoprotein (LDL), and AMD.While the exact aetiology of many such diseases requires furtherclarification, PUFAs oxidation, and consequent cross-linking orderivatisation with reactive carbonyls, often plays a prominent role.The role of oxidative stress in Age-related Macular Degeneration (AMD)is known to be quite prominent (Beatty S, et al. Survey Ophtalm. 2000;45:115-134; (de Jong Paulus T V M Age-related macular degeneration. TheNew England journal of medicine 2006; 355(14):1474-85.); Wu J, SeregardS, et al. Survey Ophtalm. 2006; 51:461-481). Almost all majorneurological diseases are known to be linked to oxidative stress. Forinstance, oxidized membrane components accelerate beta- andalpha-synuclein aggregation, associated with Alzheimer's disease (AD)and Parkinson's disease (PD) and synucleinopathies, by covalent andnoncovalent mechanisms, respectively. Reactive products of PUFAperoxidation can trigger protein misfolding in sporadic amyloiddiseases, which are the clinically most important neurological braindiseases (Bieschke J. et al, Acc. Chem. Res. 2006; 39:611-619).

Some examples of disorders involving PUFA peroxidation and reactivecompounds formed from peroxidized PUFAs include, but are not limited to:

Age-Related Macular Degeneration (AMD), Retinitis Pigmentosa (RP) andDiabetic Retinopathy (DR)

Increased oxygen levels, exposure to light and high PUFA content lead toincreased PUFA peroxidation in the eye tissues. Oxidative stress plays amajor role in the pathogenesis of AMD (Beatty S, et al. Survey Ophtalm.2000; 45:115-134). Increased levels of PUFA peroxidation products suchas HNE and HHE have been reported in retina (Long E K, et al. Free Rad.Biol. Med. 2010; 49:1-8). PUFA peroxidation products play a major rolein formation of retinal pigment epithelial (RPE) lipofuscin, whichitself can generate ROS upon irradiation with visible light, and plays amajor role in etiology of AMD (Katz M L, Arch. Gerontol. Geriatr. 2002;34:359-370). PUFA peroxidation products, including MDA, play such aprominent role in lens pathologies including formation of cataracts,that the PUFA peroxidation was proclaimed to be an initiating step inthe human cataract pathogenesis (Borchman D. et al, J. Lipid Res. 2010;51:2473-2488). Equally important is the role of PUFA peroxidationproducts in pathophysiology of diseases of human cornea, includingpterygium and keratoconus (Shoham A, et al. Free Rad. Biol. Med. 2008;45.1047-1055). Diabetic retinopathy is also associated with oxidativestress and PUFA peroxidation (Baynes J W, Thorpe S R. Diabetes 1999;48:1-9).

In some aspects, identification of a subject who has or is susceptibleto AMD, RP or DR may be determined by diagnostic tests known in the artsuch as fluorescein angiography or by identifying abnormalities invascular processes. In addition, Optial Coherence Tomography diagnosticsmay be used to identify such subjects.

Alzheimer's Disease (AD) and Mild Cognitive Impairment (MCI)

See Cooper J L. Drugs & Aging 2003; 20:399-418. Amyloid plaques andneurofibrillary tangles are the neuropathological hallmarks of AD,although whether they are the cause or the product of the disease isstill debatable. Oxidative stress, and a related inflammation, isimplicated in the AD process. The direct evidence supporting increasedoxidative stress in AD is: (1) increased ROS-stimulating Fe, Al, and Hgin AD brain; (2) increased PUFA peroxidation and decreased PUFAs in theAD brain, and increased 4-HNE in AD ventricular fluid; (3) increasedprotein and DNA oxidation in the AD brain; (4) diminished energymetabolism and decreased cytochrome c oxidase in the brain in AD; (5)advanced glycation end products (AGE), MDA, carbonyls, peroxynitrite,heme oxygenase-1 and SOD-1 in neurofibrillary tangles and AGE, hemeoxygenase-1, SOD-1 in senile plaques; and (6) studies showing thatamyloid beta peptide is capable of generating ROS (Markesbery W R. FreeRad. Biol. Med. 1997; 23:134-147).

The abnormalities of lipid metabolism play a prominent role in AD. Allproteins involved in Amyloid precursor protein processing and Ab peptideproduction are integral membrane proteins. Moreover, the Ab producingc-secretase cleavage takes place in the middle of the membrane, so thelipid environment of the cleavage enzymes influences Ab production andAD pathogenesis (Hartmann T. et al, J. Neurochem. 2007; 103:159-170).Lipid peroxidation is marked by high levels of malondialdehyde,isoprostanes, and high level of protein modification by HNE and acrolein(Sayre L M, et al. Chem. Res. Toxicol. 2008; 21:172-188; Butterfield DA, et al. Biochim. Biophys. Acta 2010; 1801:924-929). Dietary PUFAs arethe principal risk factor for the development of late-onset sporadic AD.The degree of saturation of PUFAs and the position of the first doublebond are the most critical factors determining the risk of AD, withunsaturated fats and n-3 double bonds conferring protection and anoverabundance of saturated fats or n-6 double bonds increasing the risk.DHA and AA are particularly relevant to AD (Luzon-Toro B, et al. Neurol.Psychiatr. Brain Res. 2004; 11:149-160). DHA is the major component ofexcitable membranes, promotes maturation in infants and is a potentneuroprotective agent in the adult brain, with a potential role in theprevention of AD. AA is an important provider of eicosanoids, acting asa second messenger in many neurotransmitter systems. The interaction ofdietary PUFAs and apolipoprotein E isoforms may determine the risk andrate of sustained autoperoxidation within cellular membranes and theefficacy of membrane repair.

It has been reported that lipid peroxidation is present in the brain ofMCI patients. Several studies established oxidative damage as an earlyevent in the pathogenesis of AD, that can serve as a therapeutic targetto slow the progression or perhaps the onset of the disease. (MarkesberyW R. Arch. Neurol. 2007; 64:954-956). MCI can also be characterized byelevated levels of conjugates formed by lipid peroxidation products suchas MDA, HNE, acrolein and isoprostanes (Butterfield D A, et al. Biochim.Biophys. Acta 2010; 1801:924-929).

Identifying subjects with Alzheimer's disease or susceptible toAlzheimer's disease are known in the art. For instance, subjects may beidentified using criteria set forth by the National Institute ofNeurological and Communicative Disorders and Stroke (NINCDS)-Alzheimer'sDisease an Related Disorders Association (ADRDA). The criteria arerelated to memory, language, perceptual skills, attention, constructiveabilities, orientation, problem solving and functional abilities.Similar diagnostic tests may be used to identify MCI patients.

Amyotrophic Lateral Sclerosis (ALS)

ALS is a late-onset progressive neurodegenerative disease affectingmotor neurons (loss of upper and lower motor neurons), culminating inmuscle wasting and death from respiratory failure (Boillee S. et al,Neuron 2006; 52:39-59). The etiology of most ALS cases remains unknown;however, it is recognized that ALS is strongly associated with oxidativestress. Familial ALS (fALS) is caused by oxidation of mutated SOD(superoxide dismutase) (Kabashi E. et al, Ann. Neurol. 2007;62:553-559). There are more than 100 mutations in SOD that areassociated with the fALS (Barnham K J et al, Nature Rev. Drug Discov.2004; 3:205-214). The first step is the ‘monomerisation’ of SOD, whichthen leads to the aggregation of SOD monomers, which then form aberrantS—S bonds between themselves (Kabashi E. et al, Ann. Neurol. 2007;62:553-559), yielding conglomerates which are toxic (either because theymis-fold and clog things up, or both (Barnham K J et al, Nature Rev.Drug Discov. 2004; 3:205-214).

fALS-associated SOD1 mutations were shown to be linked with the loss ofredox sensor function in NADPH oxidase-dependent ROS production, leadingto microglial neurotoxic inflammatory responses, mediated by anuncontrolled ROS generation (Liu Y, Hao W L, et al. J. Biol. Chem. 2009;284:3691-3699). Sporadic ALS (sALS) is more common (90% cases).

The aetiology of ALS cases remains unknown, but it is recognized thatALS is associated with oxidative stress and inflammation. Proteinoxidation is increased 85% in sALS patients in one study (Coyle J T. etal, Science 1993; 262:689-695). And both increased lipid peroxidationand HNE formation were reported for ALS cases, both familial andsporadic (Simpson E P et al, Neurology 2004; 62:1758-1765), in thecentral nervous system (CNS) tissue, spinal fluid, and serum. The sourceof the oxidative stress in ALS is not clear but may derive from severalprocesses including excitotoxicity, mitochondrial dysfunction, ironaccumulation or immune activation (Simpson E P et al, Neurology 2004;62:1758-1765). There is evidence that mitochondria play an importantrole in fALS and sALS, being both a trigger and a target for oxidativestress in ALS (Bacman S R et al, Molec. Neurobiol. 2006; 33:113-131).Inhibition of COX-2 has been reported to reduce spinal neurodegenerationand prolong the survival of ALS transgenic mice (Minghetti L. JNeuropathol Exp Neurol 2004; 63:901-910), highlighting the role for PUFAoxidation products in the etiology of ALS. There is also evidence ofincreased HHE-protein conjugation in ALS patients (Long E K, Picklo M J.Free Rad. Biol. Med. 2010; 49:1-8). Despite of oxidative stress beingassociated with ALS, trials of antioxidant therapies so far failed(Barber S C et al. Biochim. Biophys. Acta 2006; 1762:1051-1067).

Identifying a subject having or at risk for developing ALS may bedetermined using diagnostic methods known in the art. For example, oneor a combination of tests may be used such as upper and lower motorneuron signs in a single limb; electromyography (EMG); nerve conductionvelocity (NCV) measurement to rule out peripheral neuropathy andmyopathy; magnetic resonance imaging (MRI); and/or blood and urinetesting to eliminate a possibility of other diseases.

Other CNS diseases that may be treated by the compounds disclosed hereinare also contemplated and include degenerative neurological andneuromuscular diseases and disorders such as Jacobson Syndrome, SpinalMuscular Atrophy, and Multiple System Atrophy, among others.

Atherosclerotic Vascular Disease (ASVD)

This condition, which is a result of a build-up of fatty materialsaffecting blood vessels, results in many pathologies includingmyocardial infarction and stroke. PUFA peroxidation products play a veryimportant role in formation and accumulation of low densitylipopolyprotein (LDL, ‘bad fat’) (Esterbauer H, et al. Free Rad. Biol.Med. 1991; 11:81-128; Requena J R et al, Biochem. J. 1997; 322:317-325).Numerous diagnostic tests are available to identify subjects havingatherosclerotic vascular disease.

In some embodiments, the ratio of HDL to LDL is significantly increasedupon administration of modified PUFAs described herein. For example, inTable 3 below, an increase of approximately 86% of the HDL:LDL ratioupon administration of D-PUFA was found in comparison to the HDL:LDLratio upon administration the H-PUFA. This percentage is based upon thecalculation wherein the LDL level equals the total cholesterol minus theHDL level and minus 20% of the triglyceride level. In some aspects, theHDL-LDL ratio increases upon administration (such as over the course ofan administration protocol) of the modified PUFA at least about 5%, suchas at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%,110%, 120%, 130%, 140%, or more in comparison to the HDL:LDL ratiobefore administration.

Mitochondrial Diseases Such as Coenzyme Q10 Deficiency (010-)

Mitochondrial deficiency or mitochondrial respiration deficiencydiseases include diseases and disorders caused by oxidation ofmitochondrial membrane elements, such as mitochondrial respirationdeficiency, which occurs in the membrane. Membrane functionality isimportant to overall mitochondrial function.

Coenzyme Q deficiency is associated with many diseases, includingnervous system diseases (dyskinesias, ataxia); musculoskeletal diseases(muscle weakness, neuromuscular diseases); metabolic diseases etc. Q10plays an important role in controlling the oxidative stress. Q10—hasbeen shown to be linked to increased PUFA toxicity, through PUFAperoxidation and toxicity of the formed products (Do T Q et al, PNAS USA1996; 93:7534-7539). Numerous diagnostic tests are known in the art toidentify subjects having a Coenzyme Q10 deficiency.

Down's Syndrome (DS)

DS (trisomy of chromosome 21) is associated with premature aging andmental retardation similar to Alzheimer's disease. The incidence ofautoimmune diseases and cataracts is also elevated, pointing toincreased oxidative stress in individuals with DS (Jovanovic S V, et al.Free Rad. Biol. Med. 1998; 25:1044-1048). Chromosome 21 codes for Cu/ZnSOD and amyloid beta-peptide, so the DS is characterised by the overflowof these gene products and metabolites, notably an increased ratio ofSOD to catalase, accompanied by excessive H₂O₂ (Sinet P M. Ann. NY Acad.Sci. 1982; 396:83-94). In individuals with DS, the markers of proteinand lipid oxidation (MDA, HNE, etc), and advanced glycation andlipoxidation end-products, are significantly increased (Busciglio J,Yankner B A. Nature 1995; 378:776-779; Odetti P, et al. Biochem.Biophys. Res. Comm. 1998; 243:849-851). The importance of oxidativestress in DS led to widespread attempts to reduce the side-effect ofoxidation by employing antioxidants; but recent randomised trials foundno evidence of efficiency of antioxidant supplements (Ellis J M, et al.Brit. Med. J. 2008; 336:594-597). Subjects with Down Syndrome may beidentified by standard chromosomal testing.

Parkinson's Disease (PD)

PD is associated with oxidative stress caused by ROS, which contributesto a cascade leading to dopamine cell degeneration in PD. However,oxidative stress is intimately linked to other components of disease anddegenerative processes, such as mitochondrial dysfunction,excitotoxicity, nitric oxide toxicity and inflammation. Formation ofintracellular toxic lipid peroxides has been directly linked to damagein nigral neurons through activation of toxic cellular cascades.Oxidative damage associated with PD is initiated at the PUFAs level, andthen passed on to proteins and nuclear DNA and mtDNA (for example, insynuclein processing/Lewy body formation), and toxic carbonyl productsof oxidative damage, such as HNE and MDA, can further react withproteins to impair cell viability. Nitric oxide is known to react withsuperoxide to produce peroxynitrite and ultimately hydroxyl radical.Altered degradation of proteins has been implicated as key todopaminergic cell death in PD. Oxidative stress can impair theseprocesses directly, and products of oxidative damage, such as HNE, candamage the 26S proteasome. HNE has been directly implicated in thepathogenesis of PD (Selley M L. Free Rad. Biol. Med. 1998; 25:169-174;Zimniak P, Ageing Res. Rev. 2008; 7:281-300). Furthermore, impairment ofproteasomal function leads to free radical generation and oxidativestress (Jenner P. Annals Neurol. 2003; 53:S26-S36). An additional sourceof ROS relevant to PD etiology is dopamine (DA) turnover in dopaminergicneurons (Hastings T G, J Bioenerg. Biomembr. 2009; 41:469-72). Oxidativedamage to nucleic acids, mediated through PUFA peroxidation products,also contributes to etiology of PD (Martin L J, J. Neuropathol. Exp.Neurol. 2008; 67:377-87; Nakabeppu Y. et al., J. Neurosci. Res, 2007;85:919-34). Whether or not oxidative stress is the cause or theconsequence of PD, reducing it is likely to affect the progression ofthe disease.

Identifying a subject that has or is susceptible to Parkinson's diseasemay be determined by various tests known in the art. For example, acombination of tests and diagnosis may be based on medical history andneurological examination, including, for example, positive response tolevodopa. In addition, the identification of a subject may be determinedaccording to diagnostic criteria of Parkinson's Disease Society BrainBank and the National Institute of Neurological Disorders and Stroke,such as bradykinesia and rigidity and/or rest tremor and/or posturalinstability.

Schizophrenia and Bipolar Disorder (BD)

PUFAs are known to influence neurodevelopment and some psychiatricdisorders, such as schizophrenia. DHA, eicosapentaenoic acid (EPA) andAA are of particular importance in this regard. In schizophrenia, thereis a positive correlation between EPA supplementation and theimprovement of some symptoms, (Luzon-Toro B, et al. Neurol. Psychiatr.Brain Res. 2004; 11:149-160). There is a significant increase inoxidative stress and HNE levels in both Schizophrenia and BD (Wang J F,et al. Bipolar Disorders 2009; 11:523-529). Synaptic dysfunction isknown to be an early pathogenic event in neuropathologies such as AD,ALS, PD, etc. (LoPachin R M et al Neurotoxicol. 2008; 29:871-882).Although the molecular mechanism of this synaptotoxicity is not known,published evidence suggests that these diseases are characterized by acommon pathophysiological cascade involving oxidative stress, PUFAperoxidation (FIG. 1) and the subsequent liberation of α,β-unsaturatedcarbonyl derivatives such as acrolein and 4-HNE.

Numerous diagnostic tests are known in the art to identify subjectshaving schizophrenia or bipolar disorder.

The latest research suggests that the strongest detrimental effect onthe aetiology of oxidative stress-related diseases, includingneurological disorders, is exercised not by oxidative stress or ROS, butspecifically by electrophilic toxicity of reactive carbonyl compounds(Zimniak P, Ageing Res. Rev. 2008; 7:281-300). These carbonyl compoundscan cause nerve terminal damage by forming adducts with presynapticproteins. Therefore, the endogenous generation of acrolein and HNE inoxidatively stressed neurons of certain brain regions is mechanisticallyrelated to the synaptotoxicity associated with neurodegenerativeconditions.

In addition, acrolein and HNE are members of a large class ofstructurally related chemicals known as the type-2 alkenes. Chemicals inthis class (e.g., acrylamide, methylvinyl ketone, and methyl acrylate)are pervasive pollutants in human environments and new research hasshown that these α,β-unsaturated carbonyl derivatives are also toxic tonerve terminals. Regional synaptotoxicity, which develops during theearly stages of many neurodegenerative diseases, is mediated byendogenous generation of reactive carbonyl compounds from oxidisedPUFAs. Moreover, the onset and progression of this neuropathogenicprocess is accelerated by environmental exposure to other type-2alkenes.

Increased concentrations of 4-HNE (5-10 mM) and other reactive carbonylsare involved in the pathogenesis of a number of degenerative diseases,and thus are widely accepted as inducers and mediators of oxidativestress (Uchida K. Prog. Lipid Res. 2003; 42:318-343). However, a normal,physiological (0.1-0.3 mM) concentration of cellular 4-HNE is requiredto modulate a wide variety of cellular processes and to activatenumerous signaling pathways (Chen Z.-H., et al. IUBMB Life 2006;58:372-373; Niki E. Free Rad. Biol. Med. 2009; 47:469-484). It istherefore desirable to decrease the concentration, but not to completelyremove, reactive carbonyls from cells.

Enzymatic oxidation of PUFAs gives rise to eicosanoids and in particularto prostanoids, which comprise several important classes of biologicalmediators. Some of these mediators, in particular those formed fromomega-6 PUFAs (prostaglandins and thromboxanes), have a strongpro-inflammatory effect and may initiate blood-clotting. Existing drugssuch as aspirin have undesirable side-effects, so development of novelapproaches to downregulate the enzymatic oxidation of PUFAs, andtherefore their formation could be desirable.

The importance of oxidation of essential PUFAs in development andprogression of many neurological and other disorders served to encouragethe development of interventions designed to reduce the oxidativestress, and the associated damages inflicted by reactive carbonyls. Suchapproaches have focused on neutralizing the oxidative species(antioxidant supplements). The success of such interventions has beenlimited. Some drawbacks of such an approach include (but are not limitedto) the following points, relevant to both small molecule and enzymaticantioxidants: (a) the near-saturating amount of antioxidants alreadypresent in living cells means that any further increase, even ifsubstantial, in the amount of antioxidants would have only incremental,if any, effect on the residual ROS levels (Zimniak P, Ageing Res. Rev.2008; 7:281-300); (b) ROS play an important role in cell signalling, theinterference with which may have a detrimental effect (Packer L, CadenasE. Free Rad. Res. 2007; 41:951-952); (c) in specific physiologicalcontexts/at specific sites, ROS have protective functions which can beattenuated by antioxidants (Salganik R I. J. Am. Coll. Nutr. 2001;20:464S-472S); (d) oxidised forms of antioxidants can themselves beharmful (Zimniak P, Ageing Res. Rev. 2008; 7:281-300); (e) moderatelevels of ROS contribute to hormetic (adaptive) upregulation ofprotective mechanisms (Calabrese E J, et al. Toxicol. Appl. Pharmacol.2007; 222:122-128); (f) reactive carbonyl compounds such as HNE and HHEare not of a free radical nature, and therefore cannot be neutralised byantioxidants. However, they are still capable of significantly alteringcellular redox status by depleting cellular sulfhydryl compounds such asglutathione (GSH).

The rate of some reactions is affected by the nature of the isotopes ofthe atoms which the bond links. In general, bonds terminating in a heavyisotope will be less liable to cleavage than a bond terminating in alighter isotope. Of particular note is that bonds between hydrogen atomsand other atoms are less liable to breakage if the hydrogen is ²H ratherthan ¹H. A similar effect is seen when comparing the rate of cleavage ofa bond between a carbon atom and another atom, where bonds with ¹³C areless liable to cleavage than bonds with ¹²C. This is known as theIsotope Effect, and is well described. Many isotopes are known to showthis effect, as is described in Isotope effects in chemical reactions.(Collins C J, Bowman N S (eds) 1970 Isotope effects in chemicalreactions).

Some aspects of this invention arise from: (1) an understanding thatwhile essential PUFAs are vital for proper functioning of lipidmembranes, and in particular of the mitochondrial membranes, theirinherent drawback, i.e., the propensity to be oxidized by ROS withdetrimental outcome, is implicated in many neurological diseases; (2)antioxidants cannot cancel the negative effects of PUFA peroxidation dueto stochastic nature of the process and the stability of PUFAperoxidation products (reactive carbonyls) to antioxidant treatment, and(3) the ROS-driven damage of oxidation-prone sites within PUFAs may beovercome by using an approach that makes them less amenable to suchoxidations, without compromising any of their beneficial physicalproperties. Some aspects of this invention describe the use of theisotope effect to achieve this, only at sites in essential PUFAs andPUFA precursors that matter most for oxidation, while other aspectscontemplate other sites in addition to those that matter most foroxidation.

It will be appreciated by those skilful in the art that the same effectcan be achieved by protecting oxidation-prone positions within PUFAsusing other chemical approaches. Certain PUFA mimetics, while possessingstructural similarity with natural PUFAs, will nevertheless be stable toROS-driven and enzymatic oxidation due to structural reinforcement.

Thus, in some embodiments, an isotopically modified polyunsaturatedfatty acid or a mimetic refers to a compound having structuralsimilarity to a naturally occurring PUFA that is stabilized chemicallyor by reinforcement with one or more isotopes, for example ¹³C and/ordeuterium. Generally, if deuterium is used for reinforcement, bothhydrogens on a methylene group may be reinforced.

Some aspects of this invention provide compounds that are analogues ofessential PUFAs with either one, several, or all bis-allylic positionssubstituted with heavy isotopes. In some embodiments, the CH₂ groups,which will become the bis-allylic position in a PUFA upon enzymaticconversion, are substituted with heavy isotopes, useful for theprevention or treatment of neurological disorders in which PUFAoxidation is a factor.

The bis-allylic position generally refers to the position of thepolyunsaturated fatty acid or mimetic thereof that corresponds to themethylene groups of 1,4-diene systems. The pro-bis-allylic positionrefers to the methylene group that becomes the bis-allylic position uponenzymatic desaturation.

In some embodiments, the chemical identity of PUFAs, i.e., the chemicalstructure without regard to the isotope substitutions or substitutionsthat mimic isotope substitutions, remains the same upon ingestion. Forinstance, the chemical identity of essential PUFAs, that is, PUFAs thatmammals such as humans do not generally synthesize, may remain identicalupon ingestion. In some cases, however, PUFAs may be furtherextended/desaturated in mammals, thus changing their chemical identityupon ingestion. Similarly with mimetics, the chemical identity mayremain unchanged or may be subject to similar extension/desaturation. Insome embodiments, PUFAs that are extended, and optionally desaturated,upon ingestion and further metabolism may be referred to as higherhomologs.

In some embodiments, naturally-occurring abundance level refers to thelevel of isotopes, for example ¹³C and/or deuterium that may beincorporated into PUFAs that would be relative to the natural abundanceof the isotope in nature. For example, ¹³C has a natural abundance ofroughly 1% ¹³C atoms in total carbon atoms. Thus, the relativepercentage of carbon having greater than the natural abundance of ¹³C inPUFAs may have greater than the natural abundance level of roughly 1% ofits total carbon atoms reinforced with ¹³C, such as 2%, but preferablygreater than 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 65%, 60%,65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% of ¹³C with respect to one ormore carbon atoms in each PUFA molecule.

Regarding hydrogen, in some embodiments, deuterium has a naturalabundance of roughly 0.0156% of all naturally occurring hydrogen in theoceans on earth. Thus, a PUFA having greater that the natural abundanceof deuterium may have greater than this level or greater than thenatural abundance level of roughly 0.0156% of its hydrogen atomsreinforced with deuterium, such as 0.02%, but preferably greater than5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 65%, 60%, 65%, 70%,75%, 80%, 85%, 90%, 95%, or 100% of deuterium with respect to one ormore hydrogen atoms in each PUFA molecule.

In some aspects, a composition of PUFAs contains both isotopicallymodified PUFAs and isotopically unmodified PUFAs. The isotopic purity isa comparison between a) the relative number of molecules of isotopicallymodified PUFAs, and b) the total molecules of both isotopically modifiedPUFAs and PUFAs with no heavy atoms. In some embodiments, the isotopicpurity refers to PUFAs that are otherwise the same except for the heavyatoms.

In some embodiments, isotopic purity refers to the percentage ofmolecules of an isotopically modified PUFAs in the composition relativeto the total number of molecules of the isotopically modified PUFAs plusPUFAs with no heavy atoms. For example, the isotopic purity may be about5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 65%, 60%, 65%, 70%,75%, 80%, 85%, 90%, 95%, or 100% of the molecules of isotopicallymodified PUFAs relative to the total number of molecules of both theisotopically modified PUFAs plus PUFAs with no heavy atoms. In someembodiments, isotopic purity of the PUFAs may be from about 50%-99% ofthe total number of molecules of the PUFAs in the composition. Twomolecules of an isotopically modified PUFA out of a total of 100 totalmolecules of isotopically modified PUFAs plus PUFAs with no heavy atoms,will have 2% isotopic purity, regardless of the number of heavy atomsthe two isotopically modified molecules contain.

In some aspects, an isotopically modified PUFA molecule may contain twodeuterium atoms, such as when the two hydrogens in a methylene group areboth replaced by deuterium, and thus may be referred to as a “D2” PUFA.Similarly, an isotopically modified PUFA molecule may contain fourdeuterium atoms and may be referred to as a “D4” PUFA.

The number of heavy atoms in a molecule, or the isotopic load, may vary.For example, a molecule with a relatively low isotopic load may contain2 or 4 deuterium atoms. In a molecule with a very high load, eachhydrogen may be replaced with a deuterium. Thus, the isotopic loadrefers to the percentage of heavy atoms in each PUFA molecule. Forexample, the isotopic load may be about 5%, 10%, 15%, 20%, 25%, 30%,35%, 40%, 45%, 50%, 65%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100%of the number of the same type of atoms in comparison to a PUFA with noheavy atoms of the same type (e.g. hydrogen would be the “same type” asdeuterium). Unintended side effects are expected to be reduced wherethere is high isotopic purity in a PUFA composition but low isotopicload in a given molecule. For example, the metabolic pathways will beless affected by using in a PUFA composition with high isotopic puritybut low isotopic load.

In some aspects, isotopically modified PUFAs impart an amount of heavyatoms in a particular tissue. Thus, in some aspects, the amount of heavymolecules will be a particular percentage of the same type of moleculesin a tissue. For example, the number of heavy molecules may be about1%-100% of the total amount of the same type of molecules. In someaspects, 10-50% the molecules are substituted with the same type ofheavy molecules.

In some embodiments, a compound with the same chemical bonding structureas an essential PUFA but with a different isotopic composition atparticular positions will have significantly and usefully differentchemical properties from the unsubstituted compound. The particularpositions with respect to oxidation, such as enzymatic oxidation oroxidation by ROS, comprise bis-allylic positions of essentialpolyunsaturated fatty acids and their derivatives, as shown in FIG. 1.The essential PUFAs isotope reinforced at bis-allylic positions shownbelow will be more stable to the oxidation. Accordingly, some aspects ofthe invention provide for particular methods of using compounds ofFormula (1), whereas the sites can be further reinforced with carbon-13.R1=alkyl or H; m=1-10; n=1-5, where at each bis-allylic position, both Yatoms are deuterium atoms, for example,

11,11-Dideutero-cis,cis-9,12-Octadecadienoic acid(11,11-Dideutero-(9Z,12Z)-9,12-Octadecadienoic acid; D2-LA); and11,11,14,14-Tetradeutero-cis,cis,cis-9,12,15-Octadecatrienoic acid(11,11,14,14-Tetradeutero-(9Z,12Z,15Z)-9,12,15-Octadecatrienoic acid;D4-ALA). In some embodiments, said positions, in addition todeuteration, can be further reinforced by carbon-13, each at levels ofisotope abundance above the naturally-occurring abundance level. Allother carbon-hydrogen bonds in the PUFA molecule may optionally containdeuterium and/or Carbon-13 at, or above, the natural abundance level.

Essential PUFAs are biochemically converted into higher homologues bydesaturation and elongation. Therefore, some sites which are notbis-allylic in the precursor PUFAs will become bis-allylic uponbiochemical transformation. Such sites then become sensitive toenzymatic oxidation or oxidation by ROS. In a further embodiment, suchpro-bis-allylic sites, in addition to existing bis-allylic sites arereinforced by isotope substitution as shown below. Accordingly, thisaspect of the invention provides for the use of compounds of Formula(2), where at each bis-allylic position, and at each pro-bis-allylicposition, both X or both Y atoms may be deuterium atoms. R1=alkyl or H;m=1-10; n=1-5; p=1-10.

Said positions, in addition to deuteration, can be further reinforced bycarbon-13, each at levels of isotope abundance above thenaturally-occurring abundance level. All other carbon-hydrogen bonds inthe PUFA molecule may contain optionally deuterium and/or carbon-13 ator above the natural abundance level.

Oxidation of PUFAs at different bis-allylic sites gives rise todifferent sets of products upon enzymatic- or ROS-driven oxidation. Forexample, 4-HNE is formed from n-6 PUFAs whereas 4-HHE is formed from n-3PUFAs (Negre-Salvayre A, et al. Brit. J. Pharmacol. 2008; 153:6-20). Theproducts of such oxidation possess different regulatory, toxic,signalling, etc. properties. It is therefore desirable to control therelative extent of such oxidations. Accordingly, some aspects of theinvention provide for the use of compounds of Formula (3),differentially reinforced with heavy stable isotopes at selectedbis-allylic or pro-bis-allylic positions, to control the relative yieldof oxidation at different sites, as shown below, such that any of thepairs of Y¹-Y^(n) and/or X¹-X^(m) at the bis-allylic or pro-bis-allylicpositions of PUFAs are Deuterium atoms. R1=alkyl or H; m=1-10; n=1-6;p=1-10

Said positions, in addition to deuteration, can be further reinforced bycarbon-13. All other carbon-hydrogen bonds in the PUFA molecule maycontain deuterium at, or above the natural abundance level. It will beappreciated that the break lines in the structure shown above representsa PUFA with a varying number of double bonds, a varying number of totalcarbons, and a varying combination of isotope reinforced bis-allylic andpro-bis-allylic sites.

Exact structures of compounds illustrated above are shown below thatprovide for both isotope reinforced n-3 (omega-3) and n-6 (omega-6)essential polyunsaturated fatty acids, and the PUFAs made from thembiochemically by desaturation/elongation, to be used to slow oxidation.The PUFAs are isotope reinforced at oxidation sensitive sites. R may beH or alkyl; * represents either ¹²C or ¹³C.

D-Linoleic acids include:

The per-deuterated linoleic acid below may be produced bymicrobiological methods, for example by growing in media containing Dand 13C.

D-Arachidonic acids include:

The per-deuterated arachidonic acid below may be produced bymicrobiological methods, such as by growing in media containing D and13C.

D-Linolenic acid include:

Per-deuterated linolenic acid below may be produced by microbiologicalmethods, such as growing in media containing D and 13C.

In some aspects of the invention, any PUFAs, whether essential or not,that are capable of being taken up from diet and used in the body, canbe utilized. In the case of essential or non-essential PUFAs orprecursors, the supplemented stabilized materials can compete with otherdietary uptake and bio-manufacture to reduce the availabledisease-causing species concentrations.

In some aspects of the invention, the PUFAs isotopically reinforced atoxidation sensitive positions as described by way of the structuresabove are heavy isotope enriched at said positions as compared to thenatural abundance of the appropriate isotope, deuterium and/orcarbon-13.

In some embodiments, the disclosed compounds are enriched to 99% isotopepurity or more. In some embodiments, the heavy isotope enrichment atsaid positions is between 50%-99% deuterium and/or carbon-13.

In a further embodiment of the invention, PUFAs or their essentialprecursors, which are isotopically reinforced at the bis-allylicpositions, are used as preventive compounds against neurologicaldiseases associated with the oxidative stress.

In a further embodiment of the invention, PUFAs or their essentialprecursors, which are isotopically reinforced at the bis-allylicpositions, or at positions which will become bis-allylic uponbiochemical desaturation, are used as preventive compounds againstneurological diseases associated with the oxidative stress.

In a further embodiment of the invention, PUFAs or their essentialprecursors, which are isotopically reinforced at the bis-allylicpositions, are used as the treatment against neurological diseasesassociated with the oxidative stress and AMD.

In a further embodiment of the invention, PUFAs or their essentialprecursors, which are isotopically reinforced at the bis-allylicpositions, or at positions which will become bis-allylic uponbiochemical desaturation, are used as the treatment against neurologicaldiseases associated with the oxidative stress and AMD.

In some embodiments, the modified fatty acids, when dosed via diet asdrugs or supplements, may be dosed as prodrugs as non-toxic andpharmaceutically suitable esters of the parent fatty acid or mimetic,such as an ethyl ester or glyceryl ester. This ester assists intolerance of the drug in the gut, assists in digestion, and relies onthe high levels of esterases in the intestines to de-esterify the esterpro-drugs into the active acid form of the drug which adsorbs. Hence, insome embodiments, the invention encompasses the pro-drug esters of themodified fatty acids herein. Examples of this type of drug in themarket, nutrition, and clinical trials literature, including Glaxo'sLovaza, (mixtures of omega 3 fatty acid esters, EPA, DHA, andalpha-linolenic acid), Abbott's Omacor (omega-3-fatty acid esters), andmost fish oil supplements (DHA and EPA esters). In some aspects,incorporation of the ester pro-drugs into tissues or cells refers to theincorporation of the modified parent PUFA as it would be used as abodily constituent.

In some embodiments, stabilized compositions mimic natural occurringfatty acids without changing their elemental composition. For example,the substituent may retain the chemical valence shell. Some embodimentsinclude naturally occurring fatty acids, mimetics, and their esterpro-drugs, that are modified chemically to be effective at preventingspecific disease mechanisms, but are modified in a way (such as isotopicsubstitution) that does not change the elemental composition of thematerial. For example, deuterium is a form of the same element hydrogen.In some aspects, these compounds maintain elemental composition and arestabilized against oxidation. Some compounds that are stabilized againstoxidation are stabilized at oxidation sensitive loci. Some compounds arestabilized against oxidation via heavy isotope substitution, then atbis-allylic carbon hydrogen bonds, etc.

In some aspects, the present composition does not include compoundsdisclosed in U.S. application Ser. No. 12/281,957.

In a further embodiment, oxidation-prone bis-allylic sites of PUFAs canbe protected against hydrogen abstraction by moving bis-allylichydrogen-activating double bonds further apart, thus eliminating thebis-allylic positions while retaining certain PUFA fluidity as shownbelow. These PUFA mimetics have no bis-allylic positions.

In a further embodiment, oxidation-prone bis-allylic sites of PUFAs canbe protected against hydrogen abstraction by using heteroatoms withvalence II, thus eliminating the bis-allylic hydrogens as shown below.These PUFA mimetics also have no bis-allylic hydrogens.

In a further embodiment, PUFA mimetics, i.e. compounds structurallysimilar to natural PUFAs but unable to get oxidized because of thestructural differences, can be employed for the above mentionedpurposes. Oxidation-prone bis-allylic sites of PUFAs can be protectedagainst hydrogen abstraction by di-methylation as shown below. ThesePUFA mimetics are dimethylated at bis-allylic sites.

In a further embodiment, oxidation-prone bis-allylic sites of PUFAs canbe protected against hydrogen abstraction by alkylation as shown below.These PUFA mimetics are dialkylated at bis-allylic sites.

In a further embodiment, cyclopropyl groups can be used instead ofdouble bonds, thus rendering the acids certain fluidity whileeliminating the bis-allylic sites as shown below. These PUFA mimeticshave cyclopropyl groups instead of double bonds.

In a further embodiment, 1,2-substituted cyclobutyl groups inappropriate conformation can be used instead of double bonds, thusrendering the acids certain fluidity while eliminating the bis-allylicsites as shown below. These PUFA mimetics have 1,2-cyclobutyl groupsinstead of double bonds.

In a modification of the previous embodiment of mimetics with1,2-cyclobutyl groups instead of double bonds, 1,3-substitutedcyclobutyl groups in appropriate conformation can be used instead ofdouble bonds, thus rendering the acids certain fluidity whileeliminating the bis-allylic sites. The following PUFA mimetics have1,3-cyclobutyl groups instead of double bonds.

Compounds in some aspects of the invention are expected to be taken upby neuronal cells and tissues under appropriate conditions, as isdescribed (Rapoport S I, et al. J. Lipid Res. 2001; 42:678-685), and sowill be useful for protecting those cells or tissues against oxidativestress.

The delivery of the reinforced PUFAs or their precursors could bethrough a modified diet. Alternatively, the reinforced PUFAs or theirprecursors can be administered as foods or food supplements, on theirown or as complexes with ‘carriers’, including, but not limited to,complexes with albumin.

Other methods of delivering the reinforced PUFAs or their precursors,such as methods typically used for drug delivery and medicationdelivery, can also be employed. These methods include, but are notlimited to, peroral delivery, topical delivery, transmucosal deliverysuch as nasal delivery, nasal delivery through cribriform plate,intravenous delivery, subcutaneous delivery, inhalation, or through eyedrops.

Targeted delivery methods and sustained release methods, including, butnot limited to, the liposome delivery method, can also be employed.

A further aspect of the invention provides for the use of a compoundaccording to Formulae (1-3) and the compounds illustrated above for thetreatment of AMD and neurological diseases with oxidative stressetiology.

It is contemplated that the isotopically modified compounds describedherein may be administered over a course of time, in which the cells andtissues of the subject will contain increasing levels of isotopicallymodified compounds over the course of time in which the compounds areadministered.

It may be unnecessary to substitute all isotopically unmodified PUFAs,such as nondeuterated PUFAs, with isotopically modified PUFAs such asdeuterated PUFAs. In some embodiments, is preferable to have sufficientisotopically modified PUFAs such as D-PUFAs in the membrane to preventunmodified PUFAs such as H—PUFAs from sustaining a chain reaction ofself-oxidation. During self-oxidation, when one PUFA oxidises, and thereis a non-oxidised PUFA in the vicinity, the non-oxidised PUFA can getoxidised by the oxidised PUFA. This may also be referred to asautooxidation. In some instances, if there is a low concentration, forexample “dilute” H-PUFAs in the membrane with D-PUFAs, this oxidationcycle may be broken due to the distance separating H-PUFAs. In someembodiments, the concentration of isotopically modified PUFAs is presentin a sufficient amount to maintain autooxidation chain reaction. Tobreak the autooxidation chain reaction, for example, 1-60%, 5-50%, or15-35% of the total molecules of the same type are in the membrane. Thismay be measured by IRMS (isotope ratio mass spectrometry).

A further aspect of the invention provides a dietary, supplementary orpharmaceutical composition of the active compounds.

Compositions containing the active ingredient may be in a form suitablefor oral use, for example, as tablets, troches, lozenges, aqueous oroily suspensions, oil-in-water emulsions, dispersible powders orgranules, emulsions, hard or soft capsules, or syrups or elixirs. Suchcompositions may contain excipients such as bulking agents,solubilization agents, taste masking agents, stabilisers, colouringagents, preservatives and other agents known to those ordinarily skilledin the art of pharmaceutical formulation. In addition, oral forms mayinclude food or food supplements containing the compounds describedherein. In some embodiments supplements can be tailor-made so that onetype of PUFA, such as omega-3 or omega-6 fatty acids can be added tofood or used as a supplement depending on the dominant fat that the foodor the subject's diet contains. Moreover, compositions can betailor-made depending on the disease to be treated. For example, an LDLrelated condition may require more D-linoleic acid because cardiolipin,which is made of linoleic acid, is oxidized. In other embodiments, suchas retinal disease and neurological/CNS conditions may require moreomega-3 fatty acids such as D-linolenic acid, because D-omega-3 fattyacids are more relevant for treating these diseases. In some aspects,when the disease is associated with HNE, then D-omega-6 fatty acidsshould be prescribed, whereas for HHE, D-omega-3 fatty acids should beprescribed.

Compositions may also be suitable for delivery by topical application,as a spray, cream, ointment, lotion, or as a component or additive to apatch, bandage or wound dressing. In addition the compound can bedelivered to the site of the disease by mechanical means, or targeted tothe site of the disease through the use of systemic targetingtechnologies such as liposomes (with or without chemical modificationthat provides them with affinity for the diseased tissue), antibodies,aptamers, lectins, or chemical ligands such as albumin, with affinityfor aspects of the diseased tissue that are less abundant or not presenton normal tissue. In some aspects, topical application of cosmetics mayinclude the use of a carrier which is an isotopically modified compoundor mimetic described herein for delivering through skin such as by apatch. Eye disorders may be treated with eyedrops.

A pharmaceutical composition may also be in a form suitable foradministration by injection. Such compositions may be in the form of asolution, a suspension or an emulsion. Such compositions may includestabilizing agents, antimicrobial agents or other materials to improvethe function of the medicament. Some aspects of the invention alsoencompass dry, dessicated or freeze-dried forms of the compounds whichcan readily be formed or reconstituted into a solution suspension oremulsion suitable for administration by injection, or for oral ortopical use. Delivery by injection may be suitable for systemicdelivery, and also local delivery such as injection into the eye fortreating disorders relating to the eye.

EXAMPLES Experimental

MALDI-TOF mass-spectra were recorded on a PE-ABI Voyager Elite delayedextraction instrument. Spectra were acquired with an acceleratingvoltage of 25 KV and 100 ms delay in the positive ion mode. Unlessotherwise specified, the 1H NMR spectra were recorded on a Varian Gemini200 MHz spectrometer. HPLC was carried out on a Waters system. Chemicalswere from Sigma-Aldrich Chemical Company (USA), Avocado researchchemicals (UK), Lancaster Synthesis Ltd (UK), and Acros Organics (FisherScientific, UK). Silica gel, TLC plates and solvents were fromBDH/Merck. IR spectra were recorded with Vertex 70 spectrometer. ¹H and¹³C NMR spectra were obtained with a Bruker AC 400 instrument at 400 and100 MHz respectively, in CDCl₃ (TMS at δ=0.00 or CHCl₃ at δ=7.26 for ¹Hand CHCl₃ at δ=77.0 for ¹³C as an internal standard).

Example 1. Synthesis of 11,11-D2-linoleic acid

1,1-Dideutero-oct-2-yn-1-ol (2)

To a solution of ethylmagnesium bromide prepared from bromoethane (100ml), 1,2-dibromoethane (1 ml) and magnesium turnings (31.2 g) in dry THF(800 ml), heptyn-1 ((1); 170 ml) was added dropwise over 30-60 min underargon. The reaction mixture was stirred for 1 h, and thendeuteroparaform (30 g) was carefully added in one portion. The reactionmixture was gently refluxed for 2 h, chilled to −10° C., and then 5-7 mlof water was slowly added. The mixture was poured into 0.5 kg slurry ofcrushed ice and 40 ml concentrated sulphuric acid and washed with 0.5 Lof hexane. The organic phase was separated, and the remaining aqueousphase was extracted with 5:1 hexane:ethyl acetate (3×300 ml). Thecombined organic fraction was washed with sat. NaCl (1×50 ml), sat.NaHCO₃, (1×50 ml), and dried over Na₂SO₄. The solvent was evaporated invacuo to yield 119.3 g (99%) of colourless oil which was used withoutfurther purification. HRMS, m/z calculated for C₈H₁₂D₂O: 128.1168;found: 128.1173. ¹H NMR (CDCl₃, δ): 2.18 (t, J=7.0, 2H), 1.57 (s, 1H),1.47 (q, J=7.0 Hz, 2H), 1.31 (m, 4H), 0.87 (t, J=7.0 Hz, 3H).

1,1-Dideutero-1-bromo-oct-2-yne (3)

To a solution of (2) (3.48 g; 27.2 mmol) and pyridine (19 ml) in drydiethyl ether (300 ml), 36 ml of PBr₃ in 35 ml diethyl ether was addeddropwise with stirring over 30 min at −15° C. under argon. The reactionmixture was allowed to gradually warm up to r.t. and then refluxed 3 hwith stirring and 1 h without stirring. The reaction mixture was thencooled down to −10° C. and 500 ml of cold water was added. When theresidue dissolved, saturated NaCl (250 ml) and hexane (250 ml) wereadded, and the organic layer was separated. The aqueous fraction waswashed with hexane (2×100 ml), and the combined organic fractions werewashed with NaCl (2×100 ml) and dried over Na₂SO₄ in presence of tracesof hydroquinone and triethylamine. The solvent was removed bydistillation at atmospheric pressure followed by rotary evaporation. Theresidue was fractionated by vacuum distillation (3 mm Hg) to give 147.4g (82% counting per deutero-paraform) of pale yellow oil. B.p. 75° C.HRMS, m/z calculated for C₈H₁₁D₂Br: 190.0324; found: 189.0301, 191.0321.¹H NMR (CDCl₃, δ): 2.23 (t, J=7.0 Hz, 2H, CH₂), 1.50 (m, 2H, CH₂), 1.33(m, 4H, CH₂), 0.89 (t, J=6.9 Hz, 3H, CH₃),

11,11-Dideutero-octadeca-9,12-diynoic acid methyl ester (5)

CuI (133 g) was quickly added to 400 ml of DMF (freshly distilled overCaH₂), followed by dry NaI (106 g), K₂CO₃ (143 g). Dec-9-ynoic acidmethyl ester ((4); 65 g) was then added in one portion, followed bybromide (3) (67 g). Additional 250 ml of DMF was used to rinse thereagents off the flask walls into the bulk of reaction mixture, whichwas then stirred for 12 h. 500 ml of saturated aqueous NH₄Cl was thenadded with stirring, followed in a few minutes by saturated aqueous NaCland then by a 5:1 mixture of hexane:EtOAc (300 ml). The mixture wasfurther stirred for 15 min and then filtered through a fine mesh Schottglass filter. The residue was washed with hexane:EtOAc mix severaltimes. The organic fraction was separated, and the aqueous phase wasadditionally extracted (3×200 ml). The combined organic fraction wasdried (Na₂SO₄), traces of hydroquinone and diphenylamine were added, andthe solvent was evaporated in vacuo. The residue was immediatelydistilled at 1 mm Hg, to give 79 g (77%) of a 165-175° C. boilingfraction. HRMS, m/z calculated for C₁₉H₂₈D₂O₂: 292.2369; found:292.2365. ¹H NMR (CDCl₃, δ): 3.67 (s, 3H, OCH₃), 2.3 (t, J=7.5 Hz, 2H,CH₂), 2.14 (t, J=7.0 Hz, 4H, CH₂), 1.63 (m, 2H, CH₂), 1.47 (m, 4H, CH₂),1.3 (m, 10H, CH₂), 0.88 (t, J=7.0 Hz, 3H, CH₃).

11,11-Dideutero-cis,cis-octadeca-9,12-dienoic acid methyl ester (6)

A suspension of nickel acetate tetrahydrate (31.5 g) in 96% EtOH (400ml) was heated with stirring to approx. 50-60° C. until the saltdissolved. The flask was flushed with hydrogen, and then 130 ml of NaBH₄solution, (prepared by a 15 min stirring of NaBH₄ suspension (7.2 g) inEtOH (170 ml) followed by filtering) was added dropwise over 20-30 minwith stirring. In 15-20 min ethylenediamine (39 ml) was added in oneportion, followed in 5 min by an addition of (5) (75 g) in EtOH (200ml). The reaction mixture was very vigorously stirred under hydrogen (1atm). The absorption of hydrogen stopped in about 2 h. To the reactionmixture, 900 ml of hexane and 55 ml of ice cold AcOH were added,followed by water (15 ml). Hexane (400 ml) was added, and the mixturewas allowed to separate. Aqueous fractions were extracted by 5:1 mix ofhexane:EtOAc. The completion of extraction was monitored by TLC. Thecombined organic phase was washed with diluted solution of H₂SO₄,followed by saturated NaHCO₃ and saturated NaCl, and then dried overNa₂SO₄. The solvent was removed at reduced pressure. Silica gel (Silicagel 60, Merck; 162 g) was added to a solution of silver nitrate (43 g)in anhydrous MeCN (360 ml), and the solvent removed on a rotavap. Theobtained impregnated silica gel was dried for 3 h at 50° C. (aspirationpump) and then 8 h on an oil pump. 30 g of this silica was used per gramof product. The reaction mixture was dissolved in a small volume ofhexane and applied to the silver-modified silica gel, and pre-washedwith a 1-3% gradient of EtOAc. When the non-polar contaminants werewashed off (control by TLC), the product was eluted with 10% EtOAc andthe solvent evaporated in vacuo to give 52 g of the title ester (6) as acolourless liquid. HRMS, m/z calculated for C₁₉H₃₂D₂O₂: 296.2682; found:296.2676. IR (CCl₄): {tilde over (v)}=1740 cm⁻¹. ¹H NMR (CDCl₃, δ): 5.32(m, 4H), 3.66 (s, 3H, OCH₃), 2.29 (t, J=7.5 Hz, 2H, CH₂), 2.02 (m, 4H,CH₂), 1.60 (m, 2H, CH₂), 1.30 (m, 14H, CH₂), 0.88 (t, J=7.0 Hz, 3H,CH₃).

11,11-Dideutero-cis,cis-octadeca-9,12-dienoic acid (7)

A solution of KOH (46 g) in water (115 ml) was added to a solution ofester (6) (46 g) in MeOH (60 ml). The reaction mixture was stirred at40-50° C. for 2 h (control by TLC) and then diluted with 200 ml ofwater. Two thirds of the solvent were removed (rotavap). Dilutedsulphuric acid was added to the residue to pH 2, followed by diethylether with a little pentane. The organic layer was separated and theaqueous layer washed with diethyl ether with a little pentane. Thecombined organic fractions were washed with saturated aqueous NaCl andthen dried over Na₂SO₄. The solvent was evaporated to give 43 g of (7)(99%). IR (CCl₄): {tilde over (v)}=1741, 1711 cm⁻¹.

Example 2. Synthesis of 11,11,14,14-D4-linolenic acid

1,1-Dideutero-pent-2-yn-1-ol (9)

But-1-yne (8) was slowly bubbled through a solution of ethylmagnesiumbromide prepared from bromoethane (100 ml) and magnesium turnings (31.3g) in dry THF (800 ml) on a bath (−5° C.). Every now and then thebubbling was stopped and the cylinder with but-1-yne was weighed tomeasure the rate of consumption. The supply of alkyne was stoppedshortly after a voluminous precipitate formed (the measured mass ofalkyne consumed was 125 g). The reaction mixture was warmed up to r.t.over 30 min, and then stirred for 15 min. The mixture was then heated upto 30° C., at which point the precipitate dissolved, and then stirred atr.t. for another 30 min. Deuteroparaform (28 g) was added in one portionand the mixture was refluxed for 3 h, forming a clear solution. It wascooled down to r.t. and poured into a mixture of crushed ice (800 g) and50 ml conc. H₂SO₄. Hexane (400 ml) was added and the organic layer wasseparated. The aqueous phase was saturated with NaCl and extracted witha 4:1 mixture of hexane:EtOAc (1 L). The completion of extractionprocess was monitored by TLC. The combined organic phases were washedwith saturated NaCl, NaHCO₃ and again NaCl, and dried over Na₂SO₄. Thesolvent was removed by distillation at the atmospheric pressure (maxvapour temperature 105° C.). The residue (70.5 g; 94%) was used withoutfurther purification. HRMS, m/z calculated for C₈H₆D₂O: 86.0699; found:86.0751. ¹H NMR (CDCl₃, δ): 2.21 (q, J=7.5 Hz, 2H, CH₂), 1.93 (br s, 1H,OH), 1.12 (t, J=7.5 Hz, 3H, CH₃). ¹³C NMR (CDCl₃, δ): 87.7, 77.6, 13.7,12.3 (signal of CD₂ is absent).

1,1-Dideutero-1-bromo-pent-2-yne (10)

To a solution of (9) (70.5 g) and pyridine (16.5 ml) in dry diethylether (280 ml), 32.3 ml of PBr₃ in 50 ml diethyl ether was addeddropwise with stirring over 30 min at −10° C. under argon. The reactionmixture was allowed to gradually warm up to r.t. over 1 h. A smallamount of hydroquinone was added, and the mixture was then refluxed for4.5 h. The reaction mixture was then cooled down to −10° C. and 350 mlof cold water was added. When the residue dissolved, saturated NaCl (350ml) and hexane (300 ml) were added, and the organic layer was separated.The aqueous fraction was washed with diethyl ether (2×150 ml), and thecombined organic fractions were washed with NaCl (2×50 ml) and driedover Na₂SO₄ in presence of traces of hydroquinone and triethylamine. Thesolvent was removed at atmospheric pressure, and then the 147-155° C.boiling fraction was distilled off. Alternatively, upon reaching 100°C., the distillation at atmospheric pressure was stopped and the productdistilled off at 77-84° C. (25 mm Hg). Yield: 107 g of clear liquid.HRMS, m/z calculated for C₅H₅D₂Br: 147.9855; found: 146.9814, 148.9835.IR (CCl₄): {tilde over (v)}=2251 cm⁻¹. ¹H NMR (CDCl₃, δ): 2.23 (q, J=7.5Hz, 2H, CH₂), 1.11 (t, J=7.5 Hz, 3H, CH₃). ¹³C NMR (CDCl₃, δ): 89.3,74.5, 13.4, 12.6 (signal of CD₂ is absent).

1,1,4,4-Tetradeutero-octa-2,5-diyn-1-ol (12)

Ethylmagnesium bromide, prepared from ethyl bromide (53 ml) andmagnesium turnings (15.8 g) in 400 ml of dry THF, was added in smallportions to 350 ml of dry THF, simultaneously with acetylene bubblingthrough this mixture (at approx. 25 L/h rate) with vigorous stirring.The Grignard reagent solution was fed to the mixture at approx. 10 mlper 2-5 min. When all ethylmagnesium bromide was added (after approx.2.5 h), acetylene was bubbled through the system for another 15 min.Deuteroparaform (17.3 g) and CuCl (0.2 g) were added under argon, andthe reaction mixture was refluxed without stirring for 2.5 h, untildeuteroparaform dissolved, to yield a solution of (11). Ethylmagnesiumbromide solution, prepared from 14.8 g magnesium and 50 ml ethyl bromidein 250 ml of dry THF, was added dropwise to the reaction mixture over 20min. When the gas emanation ceased, a condenser was attached and 250 mlof solvent were distilled off. The reaction mixture was then cooled to30° C., and CuCl (1.4 g) was added followed by a dropwise addition, over15 min, of bromide (10) (69 g). The reaction mixture was then refluxedfor 5 h, cooled slightly (a precipitate will form if cooling is toofast), and poured into a slurry of crushed ice (1-1.2 kg) and 40 mlconcentrated H₂SO₄. The mixture was washed with hexane (600 ml). Theorganic fraction was separated, and the aqueous fraction wasadditionally extracted with 5:1 hexane:EtOAc (2×400 ml). The combinedorganic fraction was washed, with saturated NaCl, followed by saturatedNaHCO₃ and NaCl. The bulk of the solvent was removed at atmosphericpressure in presence of traces of hydroquinone and triethylamine. Theresidue was flushed through 100 ml of silica gel (eluent: 7:1hexane:EtOAc). The bulk of the solvent was removed at the atmosphericpressure, and the remainder on a rotavap. 49.5 g (85%) of the titlecompound obtained was used without further purification. HRMS, m/zcalculated for C₈H₆D₄O: 126.0979; found: 126.0899. IR(CCl₄): {tilde over(v)}=3622 cm⁻¹. ¹H NMR (CDCl₃, δ): 2.16 (q, J=7.5 Hz, 2H, CH₂), 1.85 (brs, 1H, OH), 1.11 (t, J=7.5 Hz, 3H, CH₃). ¹³C NMR (CDCl₃, δ): 82.3, 80.4,78.3, 72.6, 13.7, 12.2

1,1,4,4-Tetradeutero-1-bromo-octa-2,5-diyne (13)

was synthesised as described for bromide (3); 2 ml of pyridine, 14 mlPBr₃ and 250 ml of diethyl ether was used for 54.2 g of alcohol (12).The product was purified by distillation at 4 mm Hg. Yield: 53 g (65%)of (13); b.p. 100-110° C. HRMS, m/z calculated for C₈H₅D₄Br: 188.0135;found: 187.0136, 189.0143. IR (CCl₄): {tilde over (v)}=2255 cm⁻¹. ¹H NMR(CDCl₃, δ): 2.13 (q, J=7.5 Hz, 2H, CH₂); 1.07 (t, J=7.5 Hz, 3H, CH₃).¹³C NMR (CDCl₃, δ): 82.5, 81.8, 75.0, 72.0, 13.6, 12.2.

11,11,14,14-Tetradeutero-octadeca-8,12,15-triynoic acid methyl ester(15)

was synthesised in a way similar to that described for11,11-dideutero-octadeca-9,12-diynoic acid methyl ester (5). CuI (97 g)was quickly added to 400 ml of DMF (freshly distilled over CaH₂),followed by dry NaI (77.5 g), K₂CO₃ (104.5 g). Dec-9-ynoic acid methylester ((14); 47.5 g) was then added in one portion, followed by bromide(13) (48.5 g). Additional 250 ml of DMF was used to rinse the reagentsoff the flask walls into the bulk of reaction mixture, which was thenstirred for 12 h. 500 ml of saturated aqueous NH₄Cl was then added withstirring, followed in a few minutes by saturated aqueous NaCl (300 ml)followed by a 5:1 mixture of hexane:EtOAc (300 ml). The mixture wasfurther stirred for 15 min and then filtered through a fine mesh Schottglass filter. The residue was washed with hexane:EtOAc mix severaltimes. The organic fraction was separated, and the aqueous phase wasadditionally extracted (3×200 ml). The combined organic fraction wasdried (Na₂SO₄), traces of hydroquinone and diphenylamine were added, andthe solvent was evaporated in vacuo. The residue was immediatelydistilled at 1 mm Hg, to give 45.8 g (62%) of a 173-180° C. boilingfraction. An additional crystallisation was carried out as follows. Theester (15) was dissolved in hexane (500 ml) and cooled down to −50° C.The crystals formed were washed in cold hexane. The yield of this stepis 80%. HRMS, m/z calculated for C₁₉H₂₂D₄O₂: 290.2180; found: 290.2200.¹H NMR (CDCl₃, δ): 3.66 (s, 3H, OCH₃), 2.29 (t, J=7.5 Hz, 2H, CH₂), 2.15(m, 4H, CH₂), 1.61 (m, 2H, CH₂), 1.47 (m, 2H, CH₂), 1.30 (m, 6H, CH₂),1.11 (t, J=7.5 Hz, 3H, CH₃). ¹³C NMR (CDCl₃, δ): 174.1, 82.0, 80.6,74.7, 74.6, 73.7, 73.0, 51.3, 33.9, 28.9, 28.6, 28.52, 28.49, 24.8,18.5, 13.7, 12.2.

11,11,14,14-Tetradeutero-cis,cis, cis-octadeca-8,12,15-trienoic acidmethyl ester (16)

was synthesised in a way similar to that described for11,11-Dideutero-cis,cis-octadeca-9,12-dienoic acid methyl ester (‘6’). Asuspension of nickel acetate tetrahydrate (42 g) in 96% EtOH (400 ml)was heated with stirring to approx. 50-60° C. until the salt dissolved.The flask was flushed with hydrogen, and then 130 ml of NaBH₄ solution,(prepared by a 15 min stirring of NaBH₄ suspension (7.2 g) in EtOH (170ml) followed by filtering) was added dropwise over 20-30 min withstirring. In 15-20 min ethylenediamine (52 ml) was added in one portion,followed in 5 min by an addition of (15) (73 g) in EtOH (200 ml). Thereaction mixture was very vigorously stirred under hydrogen (1 atm). Theabsorption of hydrogen stopped in about 2 h. To the reaction mixture,900 ml of hexane and 55 ml of ice cold AcOH were added, followed bywater (15 ml). Hexane (400 ml) was added, and the mixture was allowed toseparate. Aqueous fractions were extracted by 5:1 mix of hexane:EtOAc.The completion of extraction was monitored by TLC. The combined organicphase was washed with diluted solution of H₂SO₄, followed by saturatedNaHCO₃ and saturated NaCl, and then dried over Na₂SO₄. The solvent wasremoved at reduced pressure. Silica gel for purification was prepared asdescribed for (6). 30 g of this silica was used per gram of product. Thereaction mixture was dissolved in a small volume of hexane and appliedto the silver-modified silica gel, and pre-washed with a 1-5% gradientof EtOAc. When the non-polar contaminants were washed off (control byTLC), the product was eluted with 10% EtOAc and the solvent evaporatedin vacuo to give 42 g of the title ester (16) as a colourless liquid.HRMS, m/z calculated for C₁₉H₂₈D₄O₂: 296.2649; found: 296.2652. IR(CCl₄): {tilde over (v)}=1740 cm⁻¹. ¹H NMR (CDCl₃, δ): 5.4 (m, 6H,CH-double bond), 3.68 (s, 3H, OCH₃), 2.33 (t, J=7.5 Hz, 2H, CH₂), 2.09(m, 4H, CH₂), 1.62 (m, 2H, CH₂), 1.33 (m, 8H, CH₂), 0.97 (t, J=7.5 Hz,3H, CH₃). ¹³C NMR (CDCl₃, δ): 174.1, 131.9, 130.2, 128.2, 128.1, 127.7,126.9, 51.3, 34.0, 29.5, 29.04, 29.02, 27.1, 25.5, 24.9, 20.5, 14.2.

11,11,14,14-Tetradeutero-cis, cis, cis-octadeca-8,12,15-trienoic acid(17)

A solution of KOH (1.5 g, 27 mmol) in water (2.6 ml was added to asolution of ester (16) (1.00 g, 3.4 mmol) in MeOH (15 ml). The reactionmixture was stirred at 40-50° C. for 2 h (control by TLC) and thendiluted with 20 ml of water. Two thirds of the solvent were removed(rotavap). Diluted sulfuric acid was added to the residue to pH 2,followed by diethyl ether with a little pentane (50 ml). The organiclayer was separated and the aqueous layer washed with diethyl ether witha little pentane (3×30 ml). The combined organic fractions were washedwith saturated aqueous NaCl and then dried over Na₂SO₄. The solvent wasevaporated to give 0.95 g of (17) (100%). IR (CCl₄): {tilde over(v)}=1741, 1711 cm⁻¹.

Example 3. Synthesis of 14,14-D2-linolenic acid

4,4-Dideutero-octa-2,5-diyn-1-ol (19)

To a solution of ethylmagnesium bromide, prepared from ethyl bromide(9.2 ml, 123.4 mmol) and magnesium turnings (2.74 g, 112.8 mmol) in 40ml of dry THF, on an ice bath with stirring, propargyl alcohol (3.16 g,56.4 mmol) in THF (5 ml) was added dropwise over 10-15 min. The reactionmixture was allowed to warm up to r.t. and stirred for another 2 h, withoccasional warming to 40° C. To thus generated dianion, 0.13 g of CuClwas added, followed by slow (over 15 min) addition of bromide (10) (6.9g) in THF (20 ml). The reaction mixture was then stirred for 1 h at r.t.and then refluxed for 5 h. The reaction mixture was then refluxed for 5h, cooled slightly (a precipitate will form if cooling is too fast), andpoured into a slurry of crushed ice and 2.5 ml concentrated H₂SO₄. Themixture was washed with hexane (600 ml). The organic fraction wasseparated, and the aqueous fraction was additionally extracted with 5:1hexane:EtOAc. The combined organic fraction was washed, with saturatedNaCl, followed by saturated NaHCO₃ and NaCl, and dried over Na₂SO₄. Thebulk of the solvent was removed at atmospheric pressure in presence oftraces of hydroquinone and triethylamine. The product was purified by CC(hexane:EtOAc=15:1) to give 3.45 g (59%) of the product 19. HRMS, m/zcalculated for C₈H₈D₂O: 124.0855; found: 124.0849. IR (CCl₄): {tildeover (v)}=3622 cm⁻¹. ¹H NMR (CDCl₃, δ): 4.21 (m, 2H, CH₂), 2.4 (m, 1H,OH), 2.16 (q, J=7.5 Hz, 2H, CH₂), 1.11 (t, J=7.5 Hz, 3H, CH₃). ¹³C NMR(CDCl₃, δ): 82.3, 80.4, 78.3, 72.6, 51.0, 13.7, 12.2.

4,4-Dideutero-1-bromo-octa-2,5-diyne (20)

was synthesised as described for (3), except all solvent was removed ona rotavap. From 3.4 g (27 mmol) of (19), 3.9 g (75%) of the bromide (20)was obtained, which was used without further purification. HRMS, m/zcalculated for C₈H₇D₂Br: 186.0011; found: 185.0019, 187.0012. IR (CCl₄):{tilde over (v)}=2255 cm⁻¹. ¹H NMR (CDCl₃, δ): 3.88 (br s, 2H, CH₂),2.13 (q, J=7.5 Hz, 2H, CH₂), 1.07 (t, J=7.5 Hz, 3H, CH₃). ¹³C NMR(CDCl₃, δ): 82.5, 81.8, 75.0, 72.0, 14.8, 13.6, 12.2.

14,14-Dideutero-octadeca-8,12,15-triynoic acid methyl ester (21)

was synthesised as described for (5). The product obtained from 9.7 gCuI, 7.8 g NaI, 10.5 g K₂CO₃, 4.85 g of bromide (20), 4.75 g of methylester (14) and 40 ml of anhydrous DMF, was purified by CC (25:1hexane:EtOAc) to give 4.5 g (60%) of the title compound. HRMS, m/zcalculated for C₁₉H₂₄D₂O₂: 288.2056; found: 288.2046. ¹H NMR (CDCl₃, δ):3.66 (s, 3H, OCH₃), 3.12 (m, 2H, CH₂), 2.29 (t, J=7.5 Hz, 2H, CH₂), 2.15(m, 4H, CH₂), 1.61 (m, 2H, CH₂), 1.47 (m, 2H, CH₂), 1.30 (m, 6H, CH₂),1.11 (t, J=7.5 Hz, 3H, CH₃). ¹³C NMR (CDCl₃, δ): 174.1, 82.0, 80.6,74.7, 74.6, 73.7, 73.0, 51.3, 33.9, 28.9, 28.6, 28.52, 28.49, 24.8,18.5, 13.7, 12.2, 9.7.

14,14-Dideutero-cis,cis,cis-octadeca-8,12,15-trienoic acid methyl ester(22)

was synthesised as described for the linoleic acid derivative (6). For areduction of 4.5 g of (21), 2.6 g of nickel acetate tetrahydrate and 3.2ml ethylenediamine was used. The product was purified onAgNO₃-impregnated silica gel as described for (6). HRMS, m/z calculatedfor C₁₉H₃₀D₂O₂: 294.2526; found: 294.2529. IR (CCl₄): {tilde over(v)}=1740 cm⁻¹. ¹H NMR (CDCl₃, δ): 5.37 (m, 6H, CH-double bond), 3.68(s, 3H, OCH₃), 2.82 (m, 2H, CH₂), 2.33 (t, J=7.5 Hz, 2H, CH₂), 2.09 (m,4H, CH₂), 1.62 (m, 2H, CH₂), 1.33 (m, 8H, CH₂), 0.97 (t, J=7.5 Hz, 3H,CH₃). ¹³C NMR (CDCl₃, δ): 174.1, 131.9, 130.2, 128.2, 128.1, 127.7,126.9, 51.3, 34.0, 29.5, 29.1, 29.04, 29.02, 27.1, 25.5, 24.9, 20.5,14.2.

14,14-Dideutero-cis,cis,cis-octadeca-8,12,15-trienoic acid (23)

To a solution of (22) (1 g, 3.4 mmol) in MeOH (15 ml), a solution of KOH(1.5 g, 27 mmol) in water (2.6 ml) was added in one portion. Thereaction mixture was then processed as described for (7) to yield 0.94 g(99%) of the title acid. IR (CCl₄): {tilde over (v)}=1741, 1711 cm⁻¹.

Example 4. Synthesis of 11,11-D2-linolenic acid

Pent-2-yn-1-ol (24)

Butyn-1 ((8); 10.4 g) was bubbled through an ice-cold solution preparedfrom bromoethane (11.2 ml) and magnesium turnings (3.6 g) in THF (100ml). The reaction mixture was allowed to warm up to r.t. and thenstirred for 15 min. The mixture was then heated up to 30° C., at whichpoint all precipitate dissolved. The heating was removed and the mixturestirred for another 30 min, and then paraform (3 g) was added in oneportion. The reaction mixture was refluxed for 3 h (all paraformdissolved), then cooled to r.t., poured into a mixture of crushed ice(80 g) and 8 ml conc. H₂SO₄, and extracted with diethyl ether. Theorganic phase was washed with saturated NaHCO₃ and NaCl, and dried overNa₂SO₄. The solvent was removed on a rotavap, and the residue (7.56 g;90%) was used without further purification. HRMS, m/z calculated forC₅H₈O: 84.0575; found: 84.0583.

1-Bromo-pent-2-yne (25)

To a solution of (24) (11.7 g) and pyridine (2.66 ml) in dry diethylether (34 ml), 5.2 ml of PBr₃ in 5 ml diethyl ether was added dropwisewith stirring over 30 min at −10° C. under argon. The reaction mixturewas allowed to gradually warm up to r.t. over 1 h. A catalytic amount ofhydroquinone was added, and the mixture was then refluxed for 4.5 h. Thereaction mixture was then cooled down to −10° C. and 35 ml of cold waterwas added. When the residue dissolved, saturated NaCl (35 ml) anddiethyl ether (30 ml) were added, and the organic layer was separated.The aqueous fraction was washed with diethyl ether (2×15 ml), and thecombined organic fractions were washed with NaCl (2×400 ml) and driedover MgSO₄. The solvent was removed at atmospheric pressure, and thenunder reduced pressure (25 mm Hg), the 60-90° C. fraction was collected.Yield: 11.1 g (84%). HRMS, m/z calculated for C₅H₇Br: 145.9731; found:144.9750, 146.9757.

1,1-Dideutero-octa-2,5-diyn-1-ol (26)

was synthesised as described for (12) with 87% yield. HRMS, m/zcalculated for C₈H₈D₂O: 124.0855; found: 124.0868. IR (CCl₄): {tildeover (v)}=3622 cm⁻¹. ¹H NMR (CDCl₃, δ): 2.65 (m, 2H, CH₂), 2.4 (m, 1H,OH), 2.1 (q, 2H, CH₂), 1.09 (t, 3H, CH₃).

1,1-Dideutero-1-bromo-octa-2,5-diyne (27)

was synthesised as described for (3), except all solvent was removed ona rotavap. The product was purified by distillation at reduced pressure.Yield: 86% (b.p. 100-105° C. at 4 mm Hg). (HRMS, m/z calculated forC₈H₇D₂Br: 186.0011; found: 184.9948, 187.9999. IR (CCl₄): {tilde over(v)}=2255 cm⁻¹. ¹H NMR (CDCl₃, δ): 2.66 (m, 2H, CH₂), 2.1 (q, 2H, CH₂),1.09 (t, 3H, CH₃).

11,11-Dideutero-octadeca-8,12,15-triynoic acid methyl ester (28)

was synthesised as described for (5). The product obtained from 7.1 gCuI, 5.66 g NaI, 7.65 g K₂CO₃, 3.55 g of bromide (27), 3.47 g of methylester (14) and 30 ml of anhydrous DMF, was purified by CC (25:1hexane:EtOAc) to give 3.7 g of the title compound. HRMS, m/z calculatedfor C₁₉H₂₄D₂O₂: 288.2056; found: 288.2069. ¹H NMR (CDCl₃, δ): 3.7 (s,3H, OCH₃), 3.15 (br. s, 2H, CH₂), 2.35 (m, 2H, CH₂), 2.17 (m, 4H, CH₂),1.61 (m, 2H, CH₂), 1.48 (m, 2H, CH₂), 1.35 (m, 6H, CH₂), 1.11 (t, 3H,CH₃).

11, 11-Dideutero-cis,cis,cis-octadeca-8,12,15-trienoic acid methyl ester(29)

was synthesised as described for the linoleic acid derivative (6). For areduction of 3.7 g of (28), 2.16 g of nickel acetate tetrahydrate and2.62 ml ethylenediamine was used. The product was purified onAgNO₃-impregnated silica gel as described for (6) to give 1.5 g. HRMS,m/z calculated for C₁₉H₃₀D₂O₂: 294.2526; found: 294.2402. IR (CCl₄):{tilde over (v)}=1740 cm⁻¹. ¹H NMR (CDCl₃, δ): 5.37 (m, 6H, CH-doublebond), 3.6 (s, 3H, OCH₃), 2.82 (m, 2H, CH₂), 2.33 (t, o=7.5 Hz, 2H,CH₂), 2.09 (m 4H, CH₂), 1.62 (m, 2H, CH₂), 1.33 (m, 8H, CH₂), 0.97 (t,J=7.5 Hz, 3H, CH₃). ¹³C NMR (CDCl₃, δ): 174.1, 131.9, 130.2, 128.2,128.1, 127.7, 126.9, 51.3, 34.0, 29.5, 29.1, 29.04, 29.02, 27.1, 25.5,24.9, 20.5, 14.2.

11,11-Dideutero-cis,cis,cis-octadeca-8,12,15-trienoic acid (30)

To a solution of (29) (1.5 g, 5.1 mmol) in MeOH (7.5 ml), a solution ofKOH (1.5 g, 27 mmol) in water (3 ml) was added in one portion. Thereaction mixture was then processed as described for (17) to yield 0.9 gof the title acid. IR (CCl₄): {tilde over (v)}=1741, 1711 cm⁻¹. ¹H NMR(CDCl₃, δ): 11.2 (br s, 1H, COOH), 5.37 (m, 6H, CH-double bond), 2.83(m, 2H, CH₂), 2.35 (t, J=7.5 Hz, 2H, CH₂), 2.06 (m 4H, CH₂), 1.63 (m,2H, CH₂), 1.32 (m, 8H, CH₂), 0.97 (t, J=7.5 Hz, 3H, CH₃). ¹³C NMR(CDCl₃, δ): 180.4, 131.9, 130.2, 128.3, 128.1, 127.6, 127.1, 34.1, 29.5,29.1, 29.03, 28.98, 27.2, 25.5, 24.6, 20.5, 14.2.

Example 5. ¹H- and ¹³C-NMR Analysis of Deuterated PUFAs Described inExamples 1-4 (FIG. 2)

Characteristic areas of ¹H and ¹³C spectra, all values in ppm. (Panel A)Deuteration of Lin acid at pos. 11 is confirmed by the disappearance ofpeaks in ¹H and ¹³C NMR spectra. Disappearance of the peak at δ_(H)2.764 is expected due to absence of H atoms (1H NMR). Disappearance ofthe peak at δ_(C) 25.5 in is due to combination of Nuclear OverhauserEffect, and splitting of this particular carbon atom into a quintet bytwo D atoms in the deuterated form of Lin acid. (Panel B) The ¹H NMRspectrum shows that the H atoms at C11 and C14 positions ofsite-specifically deuterated αLnn coincide (δ_(H) 2.801) thusdeuteration at either site (11,11-H₂, 14,14-D₂ or 11,11-D₂, 14,14-H₂)leads to a 50% decrease in integration of this peak, while deuterationof both sites (11,11,14,14-D₄) leads to the complete disappearance ofthe peak at δ_(H) 2.801. However, ¹³C NMR experiments can clearlydistinguish between the three deuterated forms, as the observed peaksfor C11 and C14 positions are separated by a small but detectabledifference. Thus, the deuteration at either C11 or C14 positions leadsto disappearance of the peak at δ_(C) 25.68 or δ_(C) 25.60,respectively, while the deuteration at both sites leads to disappearanceof the two corresponding peaks.

Example 6. Isotope Reinforcement can Shut Down PUFA Peroxidation

Q-less yeast (coq mutants) provide an ideal system to assess in vivoautoxidation of fatty acids. Coenzyme Q (ubiquinone or Q) serves as asmall lipophilic antioxidant as well as an electron shuttle in therespiratory chain of the mitochondrial inner membrane. Ten S. cerevisiaegenes (COQ1-COQ10) are required for coenzyme Q biosynthesis andfunction, and the deletion of any results in respiratory deficiency(Tran U C, Clarke C F. Mitochondrion 2007; 7S, S62). It was shown thatthe coq yeast mutants are exquisitely sensitive to autoxidation productsof PUFAs (Do T Q et al, PNAS USA 1996; 93:7534-7539; Poon W W, Do T Q,Marbois B N, Clarke C F. Mol. Aspects Med. 1997; 18, s121). Although S.cerevisiae do not produce PUFAs (Paltauf F, Daum G. Meth. Enzymol. 1992;209:514-522), they are able to utilize PUFAs when provided exogenously,allowing their content to be manipulated (Paltauf F, Daum G. Meth.Enzymol. 1992; 209:514-522). Less than 1% of Q-less (coq2, coq3, andcoq5) yeast mutants is viable following a four hour treatment withlinolenic acid (Do T Q et al, PNAS USA 1996; 93:7534-7539; Poon W W, DoT Q, Marbois B N, Clarke C F. Mol. Aspects Med. 1997; 18, s121). Incontrast, 70% of wild-type (the parental genetic background is strainW303-1B) cells subjected to this treatment remain viable. The Q-lessyeast are also hypersensitive to other PUFAs that readily autoxidize(such as arachidonic acid), but behave the same as the wild-typeparental strain to treatment with the monounsaturated oleic acid (Do T Qet al, PNAS USA 1996; 93:7534-7539). The hypersensitivity of the Q-lessyeast mutants is not a secondary effect of the inability to respire,because cor1 or atp2 mutant yeast (lacking either the bcl complex or theATP synthase, respectively) show wild-type resistance to PUFA treatment(Do T Q et al, PNAS USA 1996; 93:7534-7539; Poon W W, Do T Q, Marbois BN, Clarke C F. Mol. Aspects Med. 1997; 18, s121).

A plate dilution assay can be used to assess PUFA sensitivity. Thisassay can be performed by spotting serial five-fold dilutions ofaliquots onto YPD plate media (FIG. 3). The sensitivity of the differentstrains can be observed by visual inspection of the density of cells ineach spot.

Treatment with linolenic acid causes dramatic loss of viability of thecoq null mutants. In stark contrast, coq mutants treated with theD4-linolenic acid were not killed, and retained viabilities similar toyeast treated with oleic acid. Quantitative colony counting revealedthat the viability of cells treated with oleic and D4-linolenic wassimilar (FIG. 4), while the viability of the coq mutants was reducedmore than 100-fold following treatment with the standard linolenic acidfor 4 h. These results indicate that isotope-reinforced linolenic acidis much more resistant to autoxidation than is the standard linolenicacid, as evidenced by the resistance of the hypersensitive coq mutantsto cell killing.

GC-MS can Detect Fatty Acids and PUFAs in Yeast Cells.

Yeast do not synthesize PUFAs, however they do incorporate exogenouslysupplied linoleic and linolenic acids (Avery S V, et al. AppliedEnviron. Microbiol. 1996; 62, 3960; Howlett N G, et al. Applied Environ.Microbiol. 1997; 63, 2971).

Therefore, it seems likely that yeast would also incorporate exogenouslysupplied D4-linolenic acid. However, it is possible that thedifferential sensitivity to linolenic and D4-linolenic might beattributed to differences in integration into the cell rather thanautoxidation. To test whether this is the case, the extent of uptake ofthis fatty acid was monitored. First the conditions of separation offatty acid methyl esters (FAME) of C18:1, C18:3, D4-18:3 and C17:0 (tobe used as an internal standard) were determined. The GC-MS chromatogramshown in FIG. 5 establishes both separation and sensitivity of detectionof these fatty acid methyl ester standards.

Wild-type yeast were harvested during log phase growth and incubated inthe presence of exogenously added fatty acid (for 0 or 4 h) in thepresence of phosphate buffer plus 0.20% dextrose, as described for thefatty acid sensitivity assay. Cells were harvested, washed twice with 10ml sterile water, and the yeast cell pellets were then processed byalkaline methanolysis as described above. The fatty acids are detectedas methylesters (FAMEs) following GC-MS with C17:0 added as an internalstandard (FIG. 6). The amounts of 18:3 and D4 detected after 4 hincubation were extrapolated from the calibration curve. These resultsindicate yeast avidly incorporate both linolenic and D4-linolenic acidduring the 4 h incubation period. Based on these results, it is obviousthat the enhanced resistance of the coq mutant yeast to treatment withD4-C18:3 is not due to lack of uptake.

D2-linolenic, 11, 11-D2-linolenic acid and 14, 14-D2-linolenic acid,were also used on this yeast model and rendered comparable protection.

Example 7. D-PUFA Mitigates Oxidative Stress and Increases Survival inRetinal Cells Implicated in AMD and Diabetic Retinopathy Pathology

Several cell types, including microvascular endothelium (MVEC), retinalpigment epithelium (RPE) and retinal neurons (retinal ganglion cells)were tested for survival in cell culture. Cells were kept in the mediumcontaining either hydrogenated (control) or deuterated D2-linoleic (ω-6;LA) and D4-linolenic (ω-3; ALA) acids (20 μM; ratio of ω-6 to ω-3: 1:1or 2:1) for 72 hrs. The incorporation of PUFAs into cells was monitoredby GC. PUFAs were shown to be readily taken up by cells according to theTable 1, showing incorporation of PUFAs into MVECs.

TABLE 1 Area unlabelled Area labelled ratio control linoleate 783929764556042 0.058 e linolenate 1488866 149411 0.100 PUFA linoleate 960268305525295 0.058 linolenate 2347729 113468 0.048 Deuterated linoleate34957060 2599969 0.074 PUFA linolenate 747128 134824 0.180

The cells were then treated with paraquat (PQ; 500 μM), a commonoxidative stress-generating compound. For survival measurement, cellswere counted using haemocytometer and trypan blue exclusion method. FIG.8 shows the survival of H- and D-PUFA treated MVEC cells after acuteintoxication by paraquat. For all cell types tested, D-PUFA hadprotective effect compared to controls, similar to that shown on FIG. 8for MVEC cells.

Example 8. Isotope Ratio Mass-Spectrometry Confirms Rapid Incorporationof D-PUFA into Phospholipid Membranes of Brain Tissues

When delivering D2-LA and D4-ALA through dietary supplementation,incorporation into animal tissues cannot be monitored by chromatographybased analytical techniques because said PUFAs can be furtherextended/desaturated in mammals, thus changing their chemical identity.We used an isotope ratio mass-spectrometry technique which allows formeasurement of the total increase in deuterium composition in lipidmembranes, thus reporting on incorporation of D2-LA, D4-ALA, and anyother PUFA derived from these two. Using this method, a substantialuptake of D-PUFA into mouse brain tissue was detected. Mice weresupplemented with D-PUFA or H-PUFA as the only PUFA source for 6 days,exposed acutely to 40 mg/kg MPTP((1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine) or saline vehicle andcontinued on the same diet for an additional 6 days. MPTP is awell-recognized model in mice of Parkinson's disease. Brains wereremoved and dissected, and homogenate samples from saline-treated micewere analyzed for deuterium content. The MS was calibrated withdifferent concentrations of D2-LA and D4-ALA and compared with H-PUFAbaselines (Table 2). Table 2 shows the isotope ratio mass spectrometrymeasurement of D-PUFA incorporation into phospholipid membranes of braintissues.

The data are expressed as a ratio: the delta of the deuterium peak areain measured samples against the levels in Vienna standard mean oceanwater (V-SMOW), an MS standard for deuterium levels, ratio of the areasof the hydrogen peaks (d D/H), in per mil (‰). D-PUFA-fed mice haddeuterium levels consistent with literature references of 3-8%incorporation per day. A higher-order PUFA concentration peaks in brainwithin 8 hours after administration of a single dose of LA and ALA, andthat LA and ALA are desaturated and elongated as needed enzymatically inthe absence of higher PUFAs.

TABLE 2 Incorporation of D-PUFA into brain tissue. Group d D/H ViennaStd Mean Ocean Water  1.0 ± 0.0 H-PUFA (LA) sample −198.8 ± 2.17 D-PUFAsample 1 1703.5 ± 36.1 D-PUFA sample 2 1838.2 ± 10.8 D-PUFA sample 31973.7 ± 6.13

Each of the three samples above contain a mixture of 1:1 ratio ofD2-linolenic acid: D4-linolenic acid.

Example 9. Toxicology Studies of Mice Supplemented with D-PUFA Reveal NoAnomalies in Major Blood Biomarkers

With a more protracted dosing paradigm (i.e. 3 weeks of dietaryreplacement), chemical analysis of blood serum of H-PUFA- andD-PUFA-supplemented mice (performed at UC Davis) revealed no differencein major biomarkers of renal function, liver function, blood lipids, etcfor H-PUFA/D-PUFA saline treated mice. In this example, D-PUFA is a 2:1mixture of D2-linoleic acid: D4-linolenic acid.

Tested parameters included measurements of triglycerides; total protein;total bilirubin; phosphorus; free fatty acids; HDL; glucose; creatine;cholesterol; calcium; blood urea nitrogen; alkaline phosphatase;albumin; aspartate aminotransferase; and others in Table 3.

TABLE 3 Alanine Aspartate Alkaline Blood Urea Mouse SampleAminotransferase Aminotransferase Albumin Phosphatase Nitrogen CalciumCholesterol ID # volume U/L U/L g/dl U/L mg/dl mg/dl mg/dl 4 100 273.03008.7 3.09 81.7 19.1 7.96 148.3 5 110 5726.7 8478.9 3.42 31.1 25.4 7.40185.1 7 100 156.0 1470.6 2.82 35.1 18.9 7.64 151.2 10 60 518.4 4653.03.02 QNS 20.1 6.78 184.0 11 70 144.0 1635.3 3.63 72.7 20.3 8.75 170.8 1314 3518.1 15669.0 QNS <0.1 31.5 QNS 166.5 14 75 216.9 2107.8 3.03 42.424.4 7.46 173.6 25 75 589.5 4707.0 3.20 18.8 18.0 5.97 193.4 27 100727.2 6015.6 2.63 <0.1 36.2 5.71 166.7 28 100 468.9 4018.5 2.93 49.321.2 6.90 164.4 29 29 1898.1 12510.0 QNS QNS 24.9 QNS 208.8 30 1002963.7 5371.2 3.38 50.3 18.2 6.29 174.7 Mean 76 1508 5289 3.17 52.6 22.87.67 168.5 D-PUFA SD 33 2225 5189 0.30 23.0 4.6 0.66 14.5 D-PUFA Mean 811329 6524 3.04 39.5 23.7 6.22 181.6 H-PUFA SD 31 1078 3428 0.33 17.9 80.51 19.0 D-PUFA High Density Non-esterified Total Total MouseCreatinine Glucose Lipoprotein Fatty Acid Phosphorus Bilirubin ProteinTriglyceride ID # mg/dl mg/dl mg/dl mEq/L mg/dl mg/dl g/dl mg/dl 4 0.189160.2 104.49 1.08 13.07 0.185 5.32 38.9 5 0.356 355.6 134.37 1.07 18.590.275 6.56 57.9 7 0.154 174.6 107.39 1.11 10.14 0.192 5.26 82.7 10 0.151136.5 138.15 1.06 QNS 0.272 6.07 46.1 11 0.179 107.9 139.86 1.18  9.330.162 5.72 33.5 13 1.126 176.4 135.09 0.99 QNS QNS QNS 31.5 14 0.17093.3 47.78 1.06 10.41 0.235 6.07 43.8 25 0.126 164.5 147.96 1.01 18.390.269 6.74 41.0 27 1.453 88.3 98.46 0.87 24.57 0.301 6.26 26.9 28 0.232224.9 50.54 1.02 14.16 0.231 5.87 49.6 29 0.111 QNS 77.58 0.20 QNS QNSQNS 27.9 30 0.225 227.4 131.04 1.17 21.42 0.349 6.28 46.7 Mean 0.332172.1 115.30 1.08 12.31 0.220 5.83 47.8 D-PUFA SD 0.357 87.0 33.21 0.06 3.78 0.048 0.50 17.7 D-PUFA Mean 0.429 176.3 101.12 0.85 19.64 0.2886.29 38 H-PUFA SD 0.575 65.5 39.40 0.38  4.44 0.050 0.36 11 D-PUFA

Example 10. Supplementation with D-PUFA Increases the Level of HDL, andDecreases the Level of LDL

Mice supplemented with D-PUFA as the only source of dietary PUFA for 3weeks have slightly elevated levels of HDL (115 mg/dl; Example 9) ascompared to the control cohort dosed with H-PUFA (101 mg/dl). The D-PUFAcohort also has lower levels of cholesterol (158 mg/dl) compared toH-PUFA control group (181 mg/dl). The LDL level, i.e., the differencebetween cholesterol level and HDL, for the H-PUFA cohort is 80 mg/dl, oralmost twice as high as compared to 43 mg/dl in the D-PUFA cohort.(D-PUFA is a 1:1 mixture of D2-linoleic acid: D4-linolenic acid.)

Example 11. Mouse MPTP Model of Parkinson's Disease: D-PUFASupplementation Protects Against Dopamine Loss

Isotopic reinforcement of PUFA at bis-allylic positions preventsoxidative stress-related injury and is thus neuroprotective. Mice werefed with either D-PUFA or H-PUFA (fat-free diet (MPBio) was supplementedwith 10% fat (saturated and monounsaturated (oleic acid), of which 10%(i.e. 1% of the total fat) was a mixture of LA:ALA (1:1), orD2-LA:D4-ALA (1:1)) for six days, and then challenged with MPTP orsaline. Neurochemical analyses revealed striking neuroprotection ofstriatal dopamine with values from D-PUFA-fed mice nearly 3-fold higher:77.8±13.1 (D-PUFA; n=4) vs. 28.3±6.3 (H-PUFA; n=3) ng/mg protein.(D-PUFA is a 1:1 mixture of D2-linolenic acid: D4-linolenic acid. Asignificant improvement in the level of the DA metabolite3,4-dihydroxyphenylacetic acid (DOPAC) was also noted in the D-PUFAgroup, as well as striatal immunoreactivity for tyrosine hydroxylase(TH) by Western blot analysis. Importantly, in saline-treated mice, atrend in increased striatal DA level (11%) was noted in the D-PUFA- vs.H-PUFA-fed cohorts (p=0.053; FIG. 9).

Example 12. Attenuation of Alpha-Cynuclein Aggregation by D-PUFASupplementation

Increased a-syn expression is capable of triggering a parkinsoniansyndrome in humans and PD-like pathology in animal models.Multiplication mutations of SNCA, the a-syn gene, that result inenhanced expression of the wild-type protein are causally associatedwith autosomal dominant parkinsonism. Increased protein levels promoteself-assembly of a-syn, with formed proteinase-K resistant aggregatecongeners and nitrated/phosphorylated forms mediating pathogeniceffects. Administration of D- vs. H-PUFA as described in the previousexamples reduces the accumulation of toxic a-syn in nigral cell bodiesfrom MPTP-exposed mice, treated with D- vs. H-PUFA (FIG. 10). D-PUFA isa 1:1 mixture of D2-linolenic acid: D4-linolenic acid.

1. A method of treating or preventing the progression of aneurodegenerative disease comprising selecting a subject that has aneurodegenerative disease or is susceptible to a neurodegenerativedisease, administering an effective amount of isotopically modifiedpolyunsaturated fatty acid, mimetic or ester pro-drug thereof to thesubject; wherein upon administration, the isotopically modifiedpolyunsaturated fatty acid, mimetic or ester pro-drug thereof isincorporated in brain and/or neuronal tissue of the subject.
 2. Themethod of claim 1, wherein the subject has Alzheimer's disease or issusceptible to Alzheimer's disease.
 3. The method of claim 1, whereinthe subject has mild cognitive impairment or is susceptible to mildcognitive impairment.
 4. The method of claim 1, wherein the subject hasParkinson's disease or is susceptible to Parkinson's disease.
 5. Themethod of claim 1, wherein the subject has schizophrenia or issusceptible to schizophrenia.
 6. The method of claim 1, wherein thesubject has a bipolar disorder or is susceptible to a bipolar disorder.7. The method of claim 1, wherein the subject has amyotrophic lateralsclerosis or is susceptible to amyotrophic lateral sclerosis.
 8. Amethod of treating or preventing the progression of an oxidative diseaseof the eye comprising selecting a subject that has an oxidative diseaseof the eye or is susceptible to an oxidative disease of the eye,administering an effective amount of at least one isotopically modifiedpolyunsaturated fatty acid, mimetic or ester pro-drug thereof to thesubject; wherein upon administration, the isotopically modifiedpolyunsaturated fatty acid, mimetic or ester pro-drug thereof isincorporated in eye tissue of the subject.
 9. The method of claim 8,wherein the subject has a retinal disease or is susceptible to a retinaldisease.
 10. The method of claim 8, wherein the subject has age relatedmacular degeneration or is susceptible to age related maculardegeneration.
 11. The method of claim 8, wherein the subject hasdiabetic retinopathy or is susceptible to diabetic retinopathy.
 12. Themethod of claim 8, wherein the subject has retinitis pigmentosa or issusceptible to retinitis pigmentosa; or wherein the subject hascataracts or is susceptible to cataracts.
 13. A method comprisingselecting a subject in need of increased levels of high-densitylipoprotein and/or decreased levels of low-density lipoprotein,administering an effective amount of isotopically modifiedpolyunsaturated fatty acid, mimetic or ester pro-drug thereof to thesubject; and wherein upon administration, the level of high-densitylipoprotein is increased and/or the level of low-density lipoprotein isdecreased.
 14. The method of claim 13, wherein the subject hasatherosclerotic vascular disease or is susceptible to atheroscleroticvascular disease.
 15. (canceled)
 16. (canceled)
 17. (canceled)
 18. Amethod of treating an inborn error of metabolism comprising selecting asubject that has an inborn error of metabolism, administering aneffective amount of isotopically modified polyunsaturated fatty acid,mimetic or ester pro-drug thereof to the subject; wherein uponadministration, the isotopically modified polyunsaturated fatty acid,mimetic or ester pro-drug thereof is incorporated in brain and/orneuronal tissue of the subject.
 19. The method of claim 18 wherein theinborn error of metabolism is Down's syndrome.
 20. A polyunsaturatedfatty acid composition comprising a pharmaceutically acceptableexcipient; and an isotopically modified polyunsaturated fatty acid orester thereof comprising at least one ¹³C or at least one deuteriumatoms at a bis-allylic position; wherein the composition has an isotopicpurity in the range of 5% to 99%.
 21. The composition of claim 20wherein the isotopically modified polyunsaturated fatty acid or esterthereof comprises 11, 11, 14, 14, D4-linolenic acid, 11, 11,D2-linolenic acid, 14, 14, D2-linolenic acid, or an ester of any of theforegoing.
 22. The composition of claim 20, wherein the isotopicallymodified polyunsaturated fatty acid or ester thereof comprises deuteriumat a pro-bis-allylic position.
 23. (canceled)
 24. The composition ofclaim 20, wherein the isotopically modified polyunsaturated fatty acidor ester thereof has an isotopic purity of from about 50%-99%.
 25. Apolyunsaturated fatty acid composition comprising naturally occurringpolyunsaturated fatty acid, or ester thereof, that are modifiedchemically to be effective at preventing specific disease mechanisms;wherein the chemical modification does not change the elementalcomposition of the naturally occurring polyunsaturated fatty acid, orester thereof; with the proviso wherein the isotopically modifiedpolyunsaturated fatty acid is not 11, 11, 14, 14, D4-linolenic acid or11, 11, D2-linoleic acid.
 26. The polyunsaturated fatty acid compositionof claim 25, wherein the naturally occurring polyunsaturated fatty acid,or ester thereof is stabilized against oxidation.
 27. Thepolyunsaturated fatty acid composition of claim 25, wherein thenaturally occurring polyunsaturated fatty acid, or ester thereof isstabilized against oxidation at oxidation sensitive loci.
 28. Thepolyunsaturated fatty acid composition of claim 27, wherein thenaturally occurring polyunsaturated fatty acid, or ester thereof isstabilized against oxidation at oxidation sensitive loci with heavyisotope substitution.
 29. The polyunsaturated fatty acid composition ofclaim 28, wherein the naturally occurring polyunsaturated fatty acid, orester thereof is stabilized against oxidation at oxidation sensitiveloci at the bis-allylic carbon hydrogen atoms.
 30. A method comprisingadministering to a subject a sufficient amount of an isotopicallymodified PUFA, wherein a cell or tissue of the subject maintains asufficient concentration of isotopically modified PUFAs to maintainautooxidation of the PUFA.
 31. The polyunsaturated fatty acidcomposition of claim 20, wherein the isotopically modifiedpolyunsaturated fatty acid ester comprises an ethyl or glyceryl ester of11,11-D2-linoleic acid; 11-D-linoleic acid; 11,11,14,14-D4-linolenicacid; 11,14-D2-linolenic acid; 11,11-D2-linolenic acid; 11-D-linolenicacid; 14-D-linolenic acid; or 14,14-D2-linolenic acid.
 32. Thepolyunsaturated fatty acid composition of claim 20, wherein theisotopically modified polyunsaturated fatty acid ester comprises anethyl ester of 11,11-D2-linoleic acid; 11-D-linoleic acid;11,11,14,14-D4-linolenic acid; 11,14-D2-linolenic acid;11,11-D2-linolenic acid; 11-D-linolenic acid; 14-D-linolenic acid; or14,14-D2-linolenic acid.
 33. The polyunsaturated fatty acid compositionof claim 20, wherein the isotopically modified polyunsaturated fattyacid ester comprises an ethyl ester of 11,11-D2-linoleic acid or11-D-linoleic acid.
 34. The polyunsaturated fatty acid composition ofclaim 20, wherein the isotopically modified polyunsaturated fatty acidor ester thereof comprises a ω-6 polyunsaturated fatty acid or esterthereof.
 35. The polyunsaturated fatty acid composition of claim 20,wherein the isotopically modified polyunsaturated fatty acid or esterthereof comprises an arachidonic acid or ester thereof.
 36. Thepolyunsaturated fatty acid composition of claim 20, wherein theisotopically modified polyunsaturated fatty acid or ester thereofcomprises an eicosapentaenoic acid or ester thereof.
 37. Thepolyunsaturated fatty acid composition of claim 20, wherein theisotopically modified polyunsaturated fatty acid or ester thereofcomprises a docosahexaenoic acid or ester thereof.
 38. Thepolyunsaturated fatty acid composition of claim 20, wherein theisotopically modified polyunsaturated fatty acid or ester thereofcomprises a combination of two or more of an arachidonic acid, aneicosapentaenoic acid, a docosahexaenoic acid, or an ester of any of theforegoing.
 39. The polyunsaturated fatty acid composition of claim 20,wherein the isotopically modified polyunsaturated fatty acid or esterthereof has the following structure:

wherein: R is H or C₃H₇, R¹ is H or alkyl, each Y is independently H orDeuterium, with at least one Y being deuterium, each X is independentlyH or Deuterium, with at least one Y being deuterium, m=1-10, n=1-5, andp=1-10.
 40. The polyunsaturated fatty acid composition of claim 20,wherein the composition has an isotopic purity in the range of 10% to95%.
 41. The polyunsaturated fatty acid composition of claim 20, whereinthe composition is in a form suitable for oral administration.